ECTOINE-PRODUCING YEAST

Abstract

The present invention relates to the field of bio-production of ectoine. There is a need in the art for ectoine production methods allowing its highly efficient synthesis and secretion. The solution proposed in the present invention is the use of a genetically modified yeast comprising many modifications as described in the present text.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to the field of bio-production of ectoine.

BACKGROUND OF THE INVENTION

[0002] Ectoine (1, 4, 5, 6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is an heterocyclic amino acid naturally produced by halophilic organisms in nature. Indeed, in order to survive in salty environments, these organisms produce ectoine as compatible solute which serves as osmotic counterweights.

[0003] Ectoine is indeed also capable of protecting nucleic acids, proteins, cell membranes as well as whole cells against denaturation caused by numerous aggressions from the external environment, such as UV radiations, heating, freezing or chemical agents, but also against denaturation due to drying (see for example Lentzen G et al. Appl Microbiol Biot. 2006; 72:623-34, and Graf R et al. Clin Dermatol. 2008; 26:326-33). As such it is used in cosmetic for skin-care.

[0004] Ectoine has moreover been found to be interesting as proteins stabilizer, cosmetic additive, PCR enhancer and drying protective agent for microorganisms.

[0005] Due to these advantageous properties, ectoine is increasingly produced through bacterial processes using in particular halophilic bacteria such as Halomonas elongata. However, these methods necessitate a high salt concentration which complexifies the process and leads to an increase of the costs involved considering the important corrosion of the equipment.

[0006] Furthermore, the production of essential amino acids such as ectoine through the biosynthetic pathways of bacteria and yeasts requires an important amount of reducing power in the form of NADPH. However, the main pathway for the metabolisation of glucose in these microorganisms, and in particular in yeasts, is glycolysis followed by fermentation which only produces NADH. Maintaining an acceptable NADPH/NADH balance within the microorganism, albeit complex, is therefore essential to optimize bio-production of ectoine while maintaining a viable recombinant microorganism.

[0007] Accordingly, there is still a need in the art for further ectoine production methods allowing its highly efficient synthesis and secretion.

SUMMARY OF THE INVENTION

[0008] The present invention accordingly relates to an ectoine-producing recombinant yeast, in the genome of which:

[0009] (A) (i) at least one nucleic acid encoding an aspartokinase is overexpressed and/or is under the control of an inducible or repressible promoter; and/or

[0010] (ii) at least one nucleic acid encoding an aspartate kinase is overexpressed and/or is under the control of an inducible or repressible promoter;

[0011] (B) at least one nucleic acid encoding an aspartate semi-aldehyde dehydrogenase and/or at least one nucleic acid encoding an aspartate semi-aldehyde dehydrogenase that can use as coenzyme both NAD and NADP is overexpressed and/or is under the control of an inducible or repressible promoter;

[0012] (C) at least one nucleic acid encoding a diaminobutyrate aminotransferase is overexpressed and/or is under the control of an inducible or repressible promoter;

[0013] (D) (i) at least one nucleic acid encoding an homoserine-O-acetyltransferase MET2 is overexpressed and/or is under the control of an inducible or repressible promoter;

[0014] (ii) at least one nucleic acid encoding an homoserine-O-acetyltransferase METX is overexpressed and/or is under the control of an inducible or repressible promoter, and/or

[0015] (iii) at least one nucleic acid encoding a diaminobutyric acid acetyltransferase is overexpressed and/or is under the control of an inducible or repressible promoter;

[0016] (E) at least one nucleic acid encoding an ectoine synthase is overexpressed and/or is under the control of an inducible or repressible promoter;

[0017] (F) (i) at least one, preferably all, endogenous nucleic acid encoding an homoserine dehydrogenase has been deleted and/or interrupted, and/or

[0018] (ii) at least one, preferably all, nucleic acid encoding an homoserine dehydrogenase is independently:

[0019] under the control of an inducible or repressible promoter;

[0020] under the control of a weak promoter; and/or

[0021] in a destabilized form.

[0022] As illustrated in the enclosed examples, the recombinant yeasts of the invention have an increased ectoine production.

[0023] Said advantageous property can be further increased by also recombining the yeast with additional modifications described here-after.

[0024] An ectoine-producing recombinant yeast can consequently advantageously be used in a method for producing ectoine as described here-after or be used for the production of ectoine.

[0025] The present invention further relates to a method for producing ectoine, said method comprising the steps of:

[0026] (a) culturing a recombinant yeast of the invention in a culture medium; and

[0027] (b) recovering the ectoine from said culture medium.

[0028] Preferably, the culture medium comprises at least a carbon source, preferably a carbon source selected from the group consisting of glucose and sucrose.

[0029] The invention also relates to the use of a recombinant yeast according to the invention for the production of ectoine.

DETAILED DESCRIPTION OF THE INVENTION

[0030] The inventors have conceived genetically modified microorganisms, and especially genetically modified yeasts, having an increased ability to produce ectoine, as compared to the parent microorganisms, and especially as compared to the parent yeasts.

[0031] These genetically modified microorganisms, including these genetically modified yeasts, are described throughout the present specification.

Definitions

[0032] The term "microorganism", as used herein, refers to a yeast which is not modified artificially. The microorganism may be "donor" if it provides genetic element to be integrated in the microorganism "acceptor" which will express this foreign genetic element or if it used as tool for genetic constructions or protein expressions. The microorganism of the invention is chosen among yeast which expresses genes for the biosynthesis of ectoine.

[0033] The term "recombinant microorganism" or "genetically modified microorganism" or "recombinant yeast" or "genetically modified yeast", as used herein, refers to a yeast genetically modified or genetically engineered. It means, according to the usual meaning of these terms, that the microorganism of the invention is not found in nature and is modified either by introduction or by deletion or by modification of genetic elements from equivalent microorganism found in nature. It can also be modified by forcing the development and evolution of new metabolic pathways by combining directed mutagenesis and evolution under specific selection pressure (see for instance WO 2004/076659).

[0034] A microorganism may be modified to express exogenous genes if these genes are introduced into the microorganism with all the elements allowing their expression in the host microorganism. A microorganism may be modified to modulate the expression level of an endogenous gene. The modification or "transformation" of microorganism, like yeast, with exogenous DNA is a routine task for those skilled in the art. In particular, a genetic modification of a microorganism according to the invention, more particularly the genetic modification(s) herein defined, may be carried out by using CRISPR-Cas systems, as described in DiCarlo et al. (Nucl. Acids Res., vol. 41, No. 7, 2013: 4336-4343).

[0035] The term "endogenous gene" means that the gene was present in the microorganism before any genetic modification, in the wild-type strain. Endogenous genes may be overexpressed by introducing heterologous sequences in addition to, or to replace endogenous regulatory elements, or by introducing one or more supplementary copies of the gene into the chromosome or a plasmid. Endogenous genes may also be modified to modulate their expression and/or activity. For example, mutations may be introduced into the coding sequence to modify the gene product or heterologous sequences may be introduced in addition to or to replace endogenous regulatory elements. Modulation of an endogenous gene may result in the up-regulation and/or enhancement of the activity of the gene product, or alternatively, in the down-regulation and/or attenuation of the activity of the endogenous gene product. Another way to enhance expression of endogenous genes is to introduce one or more supplementary copies of the gene onto the chromosome or a plasmid.

[0036] The term "exogenous gene" means that the gene was introduced into a microorganism, by means well known by the man skilled in the art, whereas this gene is not naturally occurring in the wild-type microorganism. Microorganism can express exogenous genes if these genes are introduced into the microorganism with all the elements allowing their expression in the host microorganism. Transforming microorganisms with exogenous DNA is a routine task for the man skilled in the art. Exogenous genes may be integrated into the host chromosome, or be expressed extra-chromosomally from plasmids or vectors. A variety of plasmids, which differ with respect to their origin of replication and their copy number in the cell, are all known in the art. The sequence of exogenous genes may be adapted for its expression in the host microorganism. Indeed, the man skilled in the art knows the notion of codon usage bias and how to adapt nucleic sequences for a particular codon usage bias without modifying the deduced protein.

[0037] The term "heterologous gene" means that the gene is derived from a species of microorganism different from the recipient microorganism that expresses it. It refers to a gene which is not naturally occurring in the microorganism.

[0038] In the present application, all genes are referenced with their common names and with references to their nucleotide sequences and, the case arising, to their amino acid sequences. Using the references given in accession number for known genes, those skilled in the art are able to determine the equivalent genes in other organisms, bacterial strains, yeast, fungi, mammals, plants, etc. This routine work is advantageously done using consensus sequences that can be determined by carrying out sequence alignments with genes derived from other microorganisms and designing degenerated probes to clone the corresponding gene in another organism.

[0039] The man skilled in the art knows different means to modulate, and in particular up-regulate or down-regulate, the expression of endogenous genes. For example, a way to enhance expression of, or over express, endogenous genes is to introduce one or more supplementary copies of the gene onto the chromosome or a plasmid.

[0040] Another way is to replace the endogenous promoter of a gene with a stronger promoter. These promoters may be homologous or heterologous. Promoters particularly interesting in the present invention are described in more detail elsewhere in the present specification.

[0041] The nucleic acid expression construct may further comprise 5' and/or 3' recognition sequences and/or selection markers.

[0042] The term "overexpression" means that the expression of a gene or of an enzyme is increased as compared to the non-modified microorganism. Increasing the expression of an enzyme is obtained by increasing the expression of a gene encoding said enzyme. Increasing the expression of a gene may be carried out by all techniques known by the one skilled in the art. In this regard, it may be notably cited the implementation of a strong promoter upstream the nucleic acid intended to be overexpressed or the introduction of a plurality of copies of the said nucleic acid between a promoter, especially a strong promoter, and a terminator.

[0043] The term "underexpression" means that the expression of a gene or of an enzyme is decreased as compared to the non-modified microorganism. Decreasing the expression of an enzyme is obtained by decreasing the expression of a gene encoding said enzyme. Decreasing the expression of a gene may be carried out by all techniques known by the one skilled in the art. In this regard, it may be notably cited the implementation of a weak promoter upstream the nucleic acid intended to be underexpressed. It may be also cited the implementation of a nucleic acid encoding a variant of the said enzyme that is less active than the parent enzyme or a variant of the said enzyme that is more rapidly degraded in the cell than the parent enzyme. Variants of a parent enzyme that is more rapidly degraded that the said parent enzyme encompass degron-tagged enzymes. It may also be cited the decrease of the expression of a transcription activator of the gene of interest.

[0044] The term "inducible promoter" is used to qualify a promoter whose activity is induced, i.e. increased:

[0045] in the presence of one or more particular metabolite(s). The higher the metabolite concentration in the medium, the stronger the promoter activity; or

[0046] in the presence of a low concentration, or in the absence, of one or more metabolite(s). These metabolites are different from those whose increasing presence induces the activity of the promoter. The lower the metabolite concentration in the medium, the stronger the promoter activity.

[0047] The term "repressible promoter" is used to qualify a promoter whose activity is repressed, i.e. reduced:

[0048] in the presence of one or more particular metabolite(s). The higher the metabolite concentration in the medium, the weaker the promoter activity; or

[0049] in the presence of a low concentration, or in the absence, of one or more metabolite(s). These metabolites are different from those whose increasing presence represses the activity of the promoter. The lower the metabolite concentration in the medium, the weaker the promoter activity.

[0050] A used herein, a "degron-tagged" enzyme means an enzyme comprising an added protein-degradation signal amino acid sequence that serves as a destruction signal that will cause the said enzyme to be the subject of a degradation, which may be either (i) a ubiquitin-independent degradation or (ii) an ubiquitin-dependent degradation. The said added protein-degradation signal, that is also termed "degron" in the art, encompasses an amino acid sequence that serves as a destruction signal, the said amino acid sequence consisting of a transferrable degradation signal causing a targeted protein degradation. Degrons encompass "N-degrons", which are transferrable N-terminal amino acids that cause the target protein degradation following the well-known N-end rule (Bachmair et al., 1986, Science, Vol. 234 (4773): 179-186). The unstable nature of the N-degron is attributed to its first amino acids, which are prone to acetylation or arginylation modifications and ultimately lead to ubiquitination and degradation. Generally, a degron requires at least two components to ensure targeted protein degradation: (i) a target degradation recognition tag, such as a poly-ubiquitin tag and (ii) an unstructured amino acid sequence in close proximity to the degradation recognition tag. For degron-tagging a protein, and especially herein for degron-tagging an enzyme, the one skilled in the art may refer to Yu et al. (2015, Current Opinion in Biotechnology, Vol. 36: 199-204), Cho et al. (2010, Genes & Development, Vol. 24: 438-442), or to Fortmann et al. (2015, J Mol Biol, Vol. 427 (17): 2748-2756), Ravid et al. (2008, Nat Rev Mol Cell Biol, Vol. 9(9): 679-690) and Hochstrasser (1996, Annu Rev Genet, Vol. 30: 405-439).

[0051] The "activity" of an enzyme is used interchangeably with the term "function" and designates, in the context of the invention, the capacity of an enzyme to catalyze a desired reaction.

[0052] The terms "reduced activity" or "attenuated activity" of an enzyme mean either a reduced specific catalytic activity of the protein obtained by mutation in the amino acids sequence and/or decreased concentrations of the protein in the cell obtained by mutation of the nucleotide sequence or by deletion of the cognate corresponding gene or also by degron-tagging of the protein.

[0053] The term "enhanced activity" of an enzyme designates either an increased specific catalytic activity of the enzyme, and/or an increased quantity/availability of the enzyme in the cell, obtained for example by overexpression of the gene encoding the enzyme.

[0054] The terms "encoding" or "coding" refer to the process by which a polynucleotide, through the mechanisms of transcription and translation, produces an amino-acid sequence.

[0055] The gene(s) encoding the enzyme(s) considered in the present invention can be exogenous or endogenous.

[0056] "Attenuation" of genes means that genes are expressed at an inferior rate than in the non-modified microorganism. The attenuation may be achieved by means and methods known to the man skilled in the art and contains gene deletion obtained by homologous recombination, gene attenuation by insertion of an external element into the gene or gene expression under a weak promoter. The man skilled in the art knows a variety of promoters which exhibit different strengths and which promoter to use for a weak genetic expression.

[0057] The methods implemented in the present invention preferably require the use of one or more chromosomal integration constructs for the stable introduction of a heterologous nucleotide sequence into a specific location on a chromosome or for the functional disruption of one or more target genes in a genetically modified microbial cell. In some embodiments, disruption of the target gene prevents the expression of the related functional protein. In some embodiments, disruption of the target gene results in the expression of a non-functional protein from the disrupted gene.

[0058] Parameters of chromosomal integration constructs that may be varied in the practice of the present invention include, but are not limited to, the lengths of the homologous sequences; the nucleotide sequence of the homologous sequences; the length of the integrating sequence; the nucleotide sequence of the integrating sequence; and the nucleotide sequence of the target locus. In some embodiments, an effective range for the length of each homologous sequence is 20 to 5,000 base pairs, preferentially 50 to 100 base pairs. In particular embodiments, the length of each homologous sequence is about 50 base pairs. For more information on the length of homology required for gene targeting, see D. Burke et al., Methods in yeast Genetics--A cold spring harbor laboratory course Manual (2000).

[0059] In some embodiments, (a) disrupted gene(s) in which the above-mentioned DNA construct(s) is/are intended to be inserted may advantageously comprises one or more selectable markers useful for the selection of transformed microbial cells. Preferably, said selectable marker(s) are comprised in the DNA construct(s) according to the present invention.

[0060] In some embodiments, the selectable marker is an antibiotic resistance marker. Illustrative examples of antibiotic resistance markers include, but are not limited to the, NAT1, AURl-C, HPH, DSDA, KAN<R>, and SH BLE gene products. The NAT 1 gene product from S. noursei confers resistance to nourseothricin; the AURl-C gene product from Saccharomyces cerevisiae confers resistance to Auerobasidin A (AbA); the HPH gene product of Klebsiella pneumonia confers resistance to Hygromycin B; the DSDA gene product of E. coli allows cells to grow on plates with D-serine as the sole nitrogen source; the KAN<R> gene of the Tn903 transposon confers resistance to G418; and the SH BLE gene product from Streptoalloteichus hindustanus confers resistance to Zeocin (bleomycin).

[0061] In some embodiments, the antibiotic resistance marker is deleted after the genetically modified microbial cell of the invention is isolated. The man skilled in the art is able to choose suitable marker in specific genetic context.

[0062] In some embodiments, the selectable marker rescues an auxotrophy (e.g., a nutritional auxotrophy) in the genetically modified microbial cell. In such embodiments, a parent microbial cell comprises a functional disruption in one or more gene products that function in an amino acid or nucleotide biosynthetic pathway, such as, for example, the HIS3, LEU2, LYS1, LYS2, MET 15, TRP1, ADE2, and URA3 gene products in yeast, which renders the parent microbial cell incapable of growing in media without supplementation with one or more nutrients (auxotrophic phenotype). The auxotrophic phenotype can then be rescued by transforming the parent microbial cell with a chromosomal integration encoding a functional copy of the disrupted gene product (NB: the functional copy of the gene can originate from close species, such as Kluveromyces, Candida etc.), and the genetically modified microbial cell generated can be selected for based on the loss of the auxotrophic phenotype of the parent microbial cell.

[0063] For each of the nucleic acid sequences comprising a promoter sequence, a coding sequence (e.g. an enzyme coding sequence), or a terminator sequence, reference sequences are described herein. The present description also encompasses nucleic acid sequences having specific percentages of nucleic acid identity, with a reference nucleic acid sequence.

[0064] For each or the amino acid sequences of interest, reference sequences are described herein. The present description also encompasses amino acid sequences (e.g. enzyme amino acid sequences), having specific percentages of amino acid identity, with a reference amino acid sequence.

[0065] For obvious reasons, in all the present description, a specific nucleic acid sequence or a specific amino acid sequence which complies with, respectively, the considered nucleotide or amino acid identity, should further lead to obtaining a protein (or enzyme) which displays the desired biological activity. As used herein, the "percentage of identity" between two nucleic acid sequences or between two amino acid sequences is determined by comparing both optimally aligned sequences through a comparison window.

[0066] The portion of the nucleotide or amino-acid sequence in the comparison window may thus include additions or deletions (for example "gaps") as compared to the reference sequence (which does not include these additions or these deletions) so as to obtain an optimal alignment between both sequences.

[0067] The identity percentage is calculated by determining the number of positions at which an identical nucleic base, or an identical amino-acid residue, can be noted for both compared sequences, then by dividing the number of positions at which identity can be observed between both nucleic bases, or between both amino-acid residues, by the total number of positions in the comparison window, then by multiplying the result by hundred to obtain the percentage of nucleotide identity between the two sequences or the percentage of amino acid identity between the two sequences.

[0068] The comparison of the sequence optimal alignment may be performed by a computer using known algorithms.

[0069] Most preferably, the sequence identity percentage is determined using the CLUSTAL W software (version 1.82) the parameters being set as follows: (1) CPU MODE=ClustalW mp; (2) ALIGNMENT="full"; (3) OUTPUT FORMAT="aln w/numbers"; (4) OUTPUT ORDER="aligned"; (5) COLOR ALIGNMENT="no"; (6) KTUP (word size)="default"; (7) WINDOW LENGTH="default"; (8) SCORE TYPE="percent"; (9) TOPDIAG="default"; (10) PAIRGAP="default"; (11) PHYLOGENETIC TREE/TREE TYPE="none"; (12) MATRIX="default"; (13) GAP OPEN="default"; (14) END GAPS="default"; (15) GAP EXTENSION="default"; (16) GAP DISTANCES="default"; (17) TREE TYPE="cladogram" and (18) TREE GRAP DISTANCES="hide".

[0070] The "fermentation" or "culture" is generally conducted in fermenters with an appropriate culture medium adapted to the microorganism being cultivated, containing at least one simple carbon source, and if necessary co-substrates.

[0071] Microorganisms disclosed herein may be grown in fermentation media for the production of a product from oxaloacetate. For maximal production of ectoine, the microorganism strains used as production hosts preferably have a high rate of carbohydrate utilization. These characteristics may be conferred by mutagenesis and selection, genetic engineering, or may be natural. Fermentation media, or "culture medium", for the present cells may contain at least about 10 g/L of glucose. Additional carbon substrates may include but are not limited to monosaccharides such as fructose, mannose, xylose and arabinose; oligosaccharides such as lactose maltose, galactose, or sucrose; polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate cornsteep liquor, sugar beet molasses, and barley malt. Other carbon substrates may include glycerol.

[0072] Hence, it is contemplated that the source of carbon utilized in the present invention may encompass a wide variety of carbon containing substrates and will only be limited by the choice of organism.

[0073] Although it is contemplated that all of the above-mentioned carbon substrates and mixtures thereof are suitable in the present invention, preferred carbon substrates are glucose, fructose, and sucrose, or mixtures of these with C5 sugars such as xylose and/or arabinose for microorganisms modified to use C5 sugars, and more particularly glucose.

[0074] A preferred carbon substrate is glucose.

[0075] In addition to an appropriate carbon source, fermentation media may contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for the production of the desired product.

[0076] Besides, additional genetic modifications suitable for the growth of recombinant microorganisms according to the invention may be considered.

[0077] The terms "Aerobic conditions" refers to concentrations of oxygen in the culture medium that are sufficient for an aerobic or facultative anaerobic microorganism to use di-oxygene as a terminal electron acceptor.

[0078] "Microaerobic condition" refers to a culture medium in which the concentration of oxygen is less than that in air, i.e. oxygen concentration up to 6% 02.

[0079] An "appropriate culture medium" designates a medium (e.g. a sterile, liquid medium) comprising nutrients essential or beneficial to the maintenance and/or growth of the cell such as carbon sources or carbon substrate, nitrogen sources, for example, peptone, yeast extracts, meat extracts, malt extracts, urea, ammonium sulfate, ammonium chloride, ammonium nitrate and ammonium phosphate; phosphorus sources, for example, monopotassium phosphate or dipotassium phosphate; trace elements (e.g., metal salts), for example magnesium salts, cobalt salts and/or manganese salts; as well as growth factors such as amino acids, vitamins, growth promoters, and the like. The term "carbon source" or "carbon substrate" or "source of carbon" according to the present invention denotes any source of carbon that can be used by those skilled in the art to support the normal growth of a microorganism, including hexoses (such as glucose, galactose or lactose), pentoses, monosaccharides, oligosaccharides, disaccharides (such as sucrose, cellobiose or maltose), molasses, starch or its derivatives, cellulose, hemicelluloses and combinations thereof.

General Features of Genetic Modifications Introduced According to the Invention



[0080] Genes are over expressed by two kinds of non mutually exclusive modifications:

[0081] Placing them under the control of a strong promoter; and/or

[0082] Inserting a plurality of copies of the considered gene.

[0083] All the genome modifications are inserted in yeast according to known genetic engineering techniques:

[0084] The successive genes included in a gene construct that is introduced in the yeast genome according to the invention are of the following structure:

[0085] Prom.sub.1-ORF.sub.1-term.sub.1-ORF.sub.2-gene.sub.2-term.sub.2- . . . / . . . -Prom.sub.n-ORF.sub.n-term.sub.n, wherein:

[0086] Prom1 is a sequence regulating the expression of the coding sequence ORF1,

[0087] ORF1 is a nucleic acid sequence encoding a desired protein PROT1, and especially a desired enzyme PROT1,

[0088] Term1 is a transcription terminator sequence that mediates transcriptional termination by providing signals in the newly synthesized mRNA that trigger processes which release the mRNA from the transcriptional complex, and

[0089] "1", "2", . . . / . . . "n" may or may not describe the same ORF (Open Reading Frame), promoter or terminator. The order of the genes does not matter. "n" is an integer usually ranging from 5 and 20. These constructs are inserted in one of the yeast chromosome at a controlled location. In some embodiments, the insertion site is not essential for the functionality of the inserted construct, nor for the viability of the resulting genetically modified yeast.

[0090] When the yeast is for example Saccharomyces cerevisiae, genes introduced in the yeast genome and originating from other organisms than Saccharomyces cerevisiae are generally "transcoded" (generally codon-optimized"), meaning that these genes are synthesized with an optimal codon usage for expression in S. cerevisiae. The nucleotide sequence (and not the protein sequence) of some genes from S. cerevisiae has also been modified ("transcoded") to minimize recombination with an endogenous copy of the said gene.

[0091] Genes may be deleted through standard procedures used in yeast genetic engineering. In some embodiments, the genes targeted for deletion may be interrupted by insertion of one of the above described gene constructs, or alternatively the genes targeted for deletion are replaced by a short stretch of nucleotide.

[0092] Down regulating gene expression may be obtained by disrupting the endogenous copy of the gene and replacing it with a copy of the ORF under the control of a weak promoter. A list and sequences of weak promoters is described elsewhere in the present specification.

[0093] A gene may be rendered "inducible or repressible" by deleting the endogenous copy of the gene (if necessary) and placing a new copy of the ORF under the control of an inducible or repressible promoter. An inducible or repressible promoter is a promoter which activity is modulated or controlled, i.e. either increased or decreased upon a change in the environmental conditions or external stimuli. Induction or repression may be artificially controlled, which encompasses induction or repression by abiotic factors such as chemical compounds not found naturally in the organism of interest, light, oxygen levels, heat or cold. A list and sequence of inducible or repressible promoters is described elsewhere in the present specification.

[0094] As already specified elsewhere herein, a protein may be underexpressed by destabilization by using "the degron" technology which is described in Yu et al. 2015, (Current Opinion in Biotechnology, Vol. 36: 199-204). In brief this technology consists in introducing in the protein sequence a modification that targets it for degradation. It can consist only in the two first amino acids following the principle known as the N-end rule, or a larger sequence targeting the whole protein to the ubiquitin-preoteasome degradation pathway.

Recombinant Yeast According to the Invention

[0095] The inventors have conceived recombinant microorganisms, and especially recombinant yeasts, having an increased ability of producing ectoine.

[0096] The present invention relates to recombinant yeasts having an increased ectoine production, and wherein the increased ectoine production is obtained through a plurality of alterations that have been introduced in the genome thereof, by genetic engineering methods.

[0097] This invention pertains to an ectoine-producing recombinant yeast, in the genome of which:

[0098] (A) (i) at least one nucleic acid encoding an aspartokinase HOM3 is overexpressed and/or is under the control of an inducible or repressible promoter; and/or

[0099] (ii) at least one nucleic acid encoding an aspartate kinase AK is overexpressed and/or is under the control of an inducible or repressible promoter;

[0100] (B) at least one nucleic acid encoding an aspartate semi-aldehyde dehydrogenase HOM2 and/or at least one nucleic acid encoding an aspartate semi-aldehyde dehydrogenase HOM2 that can use as coenzyme both NAD and NADP is overexpressed and/or is under the control of an inducible or repressible promoter;

[0101] (C) at least one nucleic acid encoding a diaminobutyrate aminotransferase EctB is overexpressed and/or is under the control of an inducible or repressible promoter;

[0102] (D) (i) at least one nucleic acid encoding an homoserine-O-acetyltransferase MET2 is overexpressed and/or is under the control of an inducible or repressible promoter;

[0103] (ii) at least one nucleic acid encoding an homoserine-O-acetyltransferase METX is overexpressed and/or is under the control of an inducible or repressible promoter, and/or

[0104] (iii) at least one nucleic acid encoding a diaminobutyric acid acetyltransferase EctA is overexpressed and/or is under the control of an inducible or repressible promoter;

[0105] (E) at least one nucleic acid encoding an ectoine synthase EctC is overexpressed and/or is under the control of an inducible or repressible promoter;

[0106] (F) (i) at least one, preferably all, endogenous nucleic acid encoding an homoserine dehydrogenase HOM6 has been deleted, and/or

[0107] (ii) at least one, preferably all, nucleic acid encoding an homoserine dehydrogenase HOM6 is independently:

[0108] under the control of an inducible or repressible promoter;

[0109] under the control of a weak promoter; and/or

[0110] in a destabilized form.

[0111] The inventors have found that an increased production of ectoine by yeast cells may be reached by introducing in the genome of these yeast cells a plurality of genetic alterations. As it is fully described herein, the said plurality of genetic alterations encompass an overexpression of certain genes, a controlled expression of certain other genes, as well as repression or deletion of further other genes.

[0112] The increased ectoine production by yeast cells has been reached by the inventors by optimizing the metabolism of oxaloacetate and acetyl-CoA, so as to direct the subsequent artificially modified metabolic pathway mainly towards ectoine production whereas in the same time maintaining an optimal viability of the resulting genetically modified yeast cells.

[0113] After a lengthy research time period, the present inventors have determined that a high ectoine production by yeast cells is obtained by increasing the conversion of oxaloacetate into the successive intermediate metabolites phospho-aspartyl and aspartyl-semialdehyde, and additionally enhancing the conversion of aspartyl-semialdehyde into ectoine, while, notably, maintaining a redox status and more specifically an adapted NADH/NADPH balance allowing a good viability of the resulting recombinant yeast cells. This last point is essential and represented a significant challenge for the inventors throughout their research work.

[0114] The proposed solution according to the invention unexpectedly allows maintaining a viable NADH/NADPH equilibrium in the yeast cells throughout the ectoine-production pathway through the consumption of less reducing power, the consumption of reducing power in the form of NADH rather than NADPH, and/or the production of NADH instead of NADPH.

[0115] As disclosed in detail in the present specification, the resulting recombinant yeast cells are genetically modified so as to effect an over expression and/or a controlled expression of (i) an aspartokinase-encoding gene (HOM3) and/or of (ii) an aspartate kinase-encoding gene (AK), in particular of an aspartate kinase-encoding gene (AK), preferably an over expression of an aspartate kinase gene (AK).

[0116] Further, a recombinant yeast according to the invention comprises further genetic modifications for an optimal use of the intermediate metabolite phospho-aspartyl for aspartyl-semialdehyde production, the said further genetic modifications comprising an over expression and/or the controlled expression of an aspartate semi-aldehyde dehydrogenase-encoding gene (HOM2) and/or of a gene encoding an aspartate semi-aldehyde dehydrogenase that can use as coenzyme both NADH and NADPH.

[0117] Moreover, a recombinant yeast according to the invention comprises further genetic modifications for an optimal use of the intermediate metabolite aspartyl-semialdehyde for ectoine production, the said further genetic modifications comprising (i) an over expression and/or the controlled expression of a diaminobutyrate aminotransferase gene (EctB), (ii) an over expression and/or the controlled expression of a homoserine O-acetyltransferase-encoding gene (MET2; METX) and/or of a diaminobutyric acid acetyltransferase gene (EctA), (iii) an over expression and/or the controlled expression of an ectoine synthase gene (EctC) and (iv) the under expression and/or the controlled expression of an homoserine dehydrogenase gene (HOM6).

[0118] In some embodiments of a recombinant yeast according to the invention, the said yeast comprises further genetic modifications for an optimal use of the intermediate metabolite oxaloacetate for aspartate production, the said further genetic modifications comprising (i) an over expression and/or the controlled expression of an aspartate transaminase gene (AAT2) and/or (ii) an over expression and/or the controlled expression of a glutamate dehydrogenase that converts oxo-glutarate to glutamate gene (GDH).

[0119] In some embodiments of a recombinant yeast according to the invention, the said yeast comprises further genetic modifications for an optimal secretion of the produced ectoine, the said further genetic modifications comprising (i) the under expression and/or the controlled expression of a general amino acid permease gene (AGP3), (ii) the under expression and/or the controlled expression of a branched-chain amino-acid permease 3 gene (BAP3), (iii) the under expression and/or the controlled expression of a branched-chain amino-acid permease 2 gene (BAP2), (iv) the under expression and/or the controlled expression of a general amino acid permease gene (GAP1), (v) the under expression and/or the controlled expression of a high-affinity glutamine permease gene (GNP1), (vi) the under expression and/or the controlled expression of a general amino acid permease gene (AGP1), (vii) the under expression and/or the controlled expression of a low-affinity methionine permease gene (MUP3; MUP1), (viii) the over expression and/or the controlled expression of a probable transporter gene (AQR1) and/or (ix) the over expression and/or the controlled expression of a polyamine transporter 1 gene (TPO1).

[0120] In a particular embodiment, the at least one nucleic acid encoding a general amino acid permease, a branched-chain amino-acid permease 3, a branched-chain amino-acid permease 2, a general amino acid permease GAP1, a high-affinity glutamine permease GNP1, a general amino acid permease AGP1, a low-affinity methionine permease MUP3 and a high-affinity methionine permease MUP1 are, independently, nucleic acid from a yeast, preferably from Saccharomyces cerevisiae.

[0121] A recombinant yeast according to the invention produces ectoine with a higher yield than the parent yeast which does not contain the genetic modifications described above.

[0122] A recombinant yeast according to the invention has been genetically engineered so as to promote the expression of enzymes utilizing NADH rather than NADPH, such as an appropriate glutamate dehydrogenase or an appropriate aspartate semialdehyde dehydrogenase.

[0123] In some embodiments of a recombinant yeast according to the invention, the aspartate-semialdehyde dehydrogenase that are over expressed consist of the S. cerevisiae endogenous gene that is placed under the control of strong promoters and/or of inducible or repressible promoters.

[0124] In some embodiments, the aspartate-semialdehyde dehydrogenase is preferably encoded by the S. cerevisiae HOM2 gene.

[0125] In some embodiments, the aspartate-semialdehyde dehydrogenase is most preferably encoded by a variant of the S. cerevisiae HOM2 gene, which genes codes for a mutated HOM2 protein that uses both NAD and NADP, as it is shown in the examples herein. Such gene variant is for example illustrated in the examples and is called HOM2-2. It corresponds to the S. cerevisiae HOM2 gene mutated as discussed here-under.

[0126] The nature of the mutations aiming several amino acid residues in the aspartate-semialdehyde dehydrogenase variant in order to relaxe the high selectivity of HOM2 for NADP as coenzyme and enhance the affinity of the enzyme for NAD are known to the man skilled in the art and can for example be found in Faehnle, C. R. et al., Journal of Molecular Biology 1055-1068 (2005). In particular, the mutation S39 to E39 corresponding to the replacement of the nucleotides TCT in position 115 to 117 of the nucleotide sequence by the nucleotides GAG can be mentioned.

[0127] According to the nomenclature of the amino acids well known to the man skilled in the art, S represents a Serine and E represents a Glutamic acid.

[0128] In some embodiments, the aspartokinase is most preferably encoded by the S. cerevisiae HOM3 gene, as it is shown in the examples herein.

Aspartokinase-Encoding Gene Over Expression and/or Controlled Expression

[0129] In some embodiments of a recombinant yeast according to the invention, over expression of an aspartokinase-encoding gene is obtained by inserting, at selected location(s) of the yeast genome, one or more copies of an expression cassette comprising an aspartokinase coding sequence. Aspartokinase and an aspartokinase-encoding gene that are encompassed by the invention are detailed elsewhere in the present specification.

[0130] In some of these embodiments, the said one or more copies of an expression cassette comprising an aspartokinase coding sequence comprise(s) regulatory sequences allowing a strong expression of the aspartokinase, such as a strong promoter that is functional in yeast cells.

[0131] In addition to or as an alternative to these embodiments of a recombinant yeast according to the invention, at least one aspartokinase-encoding gene can be under the control of an inducible or repressible promoter that is functional in yeast cells.

[0132] In these embodiments, a controlled expression of an aspartokinase-encoding gene can be obtained by inserting, at the location of the natural yeast aspartokinase open reading frame, an inducible regulatory sequence, such as an inducible or repressible promoter, that replaces the endogenous promoter initially present in the yeast genome at this genome location.

[0133] Without wishing to be bound by any particular theory, the inventors believe that with over expression of an aspartokinase-encoding gene, a controlled level of conversion of aspartate into aspartyl phosphate (Aspartyl-P), also termed phospho-aspartyl, is obtained that shall contribute to the high level of viability of a recombinant yeast according to the invention. The same applies when at least one aspartokinase coding sequence is under the control of an inducible or repressible promoter.

[0134] In some preferred embodiments, the said aspartokinase-encoding gene is the HOM3 gene from Saccharomyces cerevisiae, as shown in the examples herein.

[0135] In preferred embodiments, the said aspartokinase-encoding gene is placed under the control of the strong promoter pCCW12 or of the inducible or repressible promoter pCUP-1-1.

[0136] Illustratively, the aspartokinase gene may be inserted within the HOM6 gene and/or within the SAM3 gene, as it is shown in the examples herein.

Aspartate Kinase-Encoding Gene Over Expression and/or Controlled Expression

[0137] Alternatively or in complement to the over expression and/or controlled expression of an aspartokinase as discussed here-above, a recombinant yeast according to the invention can also be such that it comprises the over expression and/or controlled expression of an aspartate kinase-encoding gene.

[0138] Accordingly, in preferred embodiments of a recombinant yeast according to the invention, over expression of an aspartate kinase-encoding gene is obtained by inserting, at selected location(s) of the yeast genome, one or more copies of an expression cassette comprising a aspartate kinase coding sequence. Aspartate kinase and an aspartate kinase-encoding gene that are encompassed by the invention are detailed elsewhere in the present specification.

[0139] In some of these embodiments, the said one or more copies of an expression cassette comprising an aspartate kinase-coding sequence comprise regulatory sequences allowing a strong expression of the aspartate kinase, such as a strong promoter that is functional in yeast cells.

[0140] In addition to or as an alternative to these embodiments of a recombinant yeast according to the invention, at least one aspartate kinase-encoding gene can be under the control of an inducible or repressible promoter that is functional in yeast cells.

[0141] Without wishing to be bound by any particular theory, the inventors believe that with a controlled expression of an aspartate kinase-encoding gene, a controlled level of conversion of aspartate into aspartyl phosphate (Aspartyl-P) is obtained that shall contribute to the high level of viability of a recombinant yeast according to the invention.

[0142] In preferred embodiments, the said aspartate kinase-encoding gene is the AK gene from Bacillus subtilis, as shown in the examples herein.

[0143] In preferred embodiments, the said aspartate kinase-encoding gene is placed under the control of the inducible or repressible promoter pACU7.

[0144] Illustratively, the aspartate kinase gene may be inserted within the TRP1 gene, as it is shown in the examples herein.

Aspartate-Semialdehyde Dehydrogenase-Encoding Gene Over Expression and/or Controlled Expression

[0145] In preferred embodiments of a recombinant yeast according to the invention, over expression of an aspartate-semialdehyde dehydrogenase-encoding gene is obtained by inserting, at selected location(s) of the yeast genome, one or more copies of an expression cassette comprising an aspartate-semialdehyde dehydrogenase coding sequence. Aspartate-semialdehyde dehydrogenase and an aspartate-semialdehyde dehydrogenase-encoding gene that are encompassed by the invention are detailed elsewhere in the present specification.

[0146] In some of these embodiments, the said one or more copies of an expression cassette comprising an aspartate-semialdehyde dehydrogenase coding sequence comprise(s) regulatory sequences allowing a strong expression of the aspartate-semialdehyde dehydrogenase, such as a strong promoter that is functional in yeast cells.

[0147] In addition to or as an alternative to these embodiments of a recombinant yeast according to the invention, at least one aspartate-semialdehyde dehydrogenase-encoding gene can be under the control of an inducible or repressible promoter that is functional in yeast cells.

[0148] Without wishing to be bound by any particular theory, the inventors believe that over expression of an aspartate-semialdehyde dehydrogenase may enhance the conversion of the intermediate metabolite aspartyl phosphate (Aspartyl-P) into aspartyl-semialdehyde. The same applies when at least one aspartate-semialdehyde dehydrogenase coding sequence is under the control of an inducible or repressible promoter.

[0149] In some embodiments, the aspartate-semialdehyde dehydrogenase may be an enzyme variant that uses both NADH or NADPH for catalyzing the conversion of aspartyl phosphate (Aspartyl-P) into aspartyl-semialdehyde.

[0150] In some preferred embodiments, the said aspartate-semialdehyde dehydrogenase-encoding gene is the HOM2 gene from Saccharomyces cerevisiae, or alternatively a variant of HOM2 utilizing both NADH and NADPH as shown in the examples herein and discussed previously.

[0151] In preferred embodiments, the said aspartate semi-aldehyde dehydrogenase-encoding gene is placed under the control of the inducible or repressible promoter pACU5 or the strong promoter pCCW12.

[0152] Illustratively, the aspartate-semialdehyde dehydrogenase gene may be inserted within the HIS3 gene, and/or within the MUP3 gene, as it is shown in the examples herein.

Diaminobutyrate Aminotransferase-Encoding Gene Over Expression and/or Controlled Expression

[0153] In preferred embodiments of a recombinant yeast according to the invention, over expression of a diaminobutyrate aminotransferase-encoding gene is obtained by inserting, at selected location(s) of the yeast genome, one or more copies of an expression cassette comprising a diaminobutyrate aminotransferase coding sequence. Diaminobutyrate aminotransferase and a diaminobutyrate aminotransferase-encoding gene that are encompassed by the invention are detailed elsewhere in the present specification.

[0154] In some of these embodiments, the said one or more copies of an expression cassette comprising a diaminobutyrate aminotransferase coding sequence comprise(s) regulatory sequences allowing a strong expression of the diaminobutyrate aminotransferase, such as a strong promoter that is functional in yeast cells.

[0155] In addition to or as an alternative to these embodiments of a recombinant yeast according to the invention, at least one diaminobutyrate aminotransferase-encoding gene can be under the control of an inducible or repressible promoter that is functional in yeast cells.

[0156] Without wishing to be bound by any particular theory, the inventors believe that over expression of an diaminobutyrate aminotransferase may enhance the conversion of the intermediate metabolite aspartyl-semialdehyde into 2,4-diaminobutyrate. The same applies when at least one diaminobutyrate aminotransferase coding sequence is under the control of an inducible or repressible promoter.

[0157] In preferred embodiments, the said diaminobutyrate aminotransferase-encoding gene is the EctB gene from Pseudomonas aeruginosa, or the EctB gene from Halomonas elongata (sometimes also named Chromohalobacter salexigens), as shown in the examples herein.

[0158] In preferred embodiments, the said diaminobutyrate aminotransferase-encoding gene is placed under the control of the strong promoter pCCW12 and/or the strong promoter pTDH3.

[0159] Illustratively, the diaminobutyrate aminotransferase gene may be inserted within the HOM6 gene and/or within the SAM3 gene and/or within the MUP3 gene and/or within the URA3 gene, as it is shown in the examples herein.

Homoserine-O-Acetyltransferase-Encoding Gene Over Expression and/or Controlled Expression

[0160] In preferred embodiments of a recombinant yeast according to the invention, over expression of a homoserine-O-acetyltransferase-encoding gene is obtained by inserting, at selected location(s) of the yeast genome, one or more copies of an expression cassette comprising a homoserine-O-acetyltransferase coding sequence. Homoserine-O-acetyltransferase and a homoserine-O-acetyltransferase-encoding gene that are encompassed by the invention are detailed elsewhere in the present specification.

[0161] In some of these embodiments, the said one or more copies of an expression cassette comprising a homoserine-O-acetyltransferase coding sequence comprise regulatory sequences allowing a strong expression of the homoserine-O-acetyltransferase, such as a strong promoter that is functional in yeast cells.

[0162] In addition to or as an alternative to these embodiments of a recombinant yeast according to the invention, at least one homoserine-O-acetyltransferase-encoding gene can be under the control of an inducible or repressible promoter that is functional in yeast cells.

[0163] Without wishing to be bound by any particular theory, the inventors believe that over expression of a homoserine-O-acetyltransferase allows, and consequently increases, the level of conversion of the intermediate metabolite 2,4-diaminobutyrate into acetyl-2,4-diaminobutyrate, in the presence of acetyl-CoA. The same applies when at least one homoserine-O-acetyltransferase coding sequence is under the control of an inducible or repressible promoter.

[0164] In preferred embodiments, the said homoserine-O-acetyltransferase-encoding gene is the MET2 gene from Saccharomyces cerevisiae, as shown in the examples herein.

[0165] In preferred embodiments, the said homoserine-O-acetyltransferase-encoding gene is the METX gene from Corynebacterium glutamicum, as shown in the examples herein.

[0166] In a particularly preferred embodiment, a recombinant yeast according to the invention comprises at least one homoserine-O-acetyltransferase-encoding gene which is the MET2 gene from Saccharomyces cerevisiae and at least one homoserine-O-acetyltransferase-encoding gene which is the METX gene from Corynebacterium glutamicum.

[0167] In preferred embodiments, the said homoserine-O-acetyltransferase-encoding gene is, independently for each copy of said gene if multiple copies are present, placed under the control of a strong promoter pPDC1 or the inducible or repressible promoter pACU6.

[0168] Illustratively, the homoserine-O-acetyltransferase gene may be inserted within the SAM3 gene and/or within the HIS3 gene, as it is shown in the examples herein.

Diaminobutyric Acid Acetyltransferase-Encoding Gene Over Expression and/or Controlled Expression

[0169] In preferred embodiments of a recombinant yeast according to the invention, over expression of a diaminobutyric acid acetyltransferase-encoding gene is obtained by inserting, at selected location(s) of the yeast genome, one or more copies of an expression cassette comprising a diaminobutyric acid acetyltransferase coding sequence. A diaminobutyric acid acetyltransferase and a diaminobutyric acid acetyltransferase-encoding gene that are encompassed by the invention are detailed elsewhere in the present specification.

[0170] In some of these embodiments, the said one or more copies of an expression cassette comprising a diaminobutyric acid acetyltransferase coding sequence comprise(s) regulatory sequences allowing a strong expression of the diaminobutyric acid acetyltransferase, such as a strong promoter that is functional in yeast cells.

[0171] In addition to or as an alternative to these embodiments of a recombinant yeast according to the invention, at least one diaminobutyric acid acetyltransferase-encoding gene can be under the control of an inducible or repressible promoter that is functional in yeast cells.

[0172] Without wishing to be bound by any particular theory, the inventors believe that over expression of a diaminobutyric acid acetyltransferase allows, and consequently increases, the level of conversion of the intermediate metabolite 2,4-diaminobutyrate into acetyl-2,4-diaminobutyrate, in the presence of acetyl-CoA. The same applies when at least one diaminobutyric acid acetyltransferase coding sequence is under the control of an inducible or repressible promoter.

[0173] In preferred embodiments, the said diaminobutyric acid acetyltransferase-encoding gene is the EctA gene from Halomonas elongata (sometimes also named Chromohalobacter salexigens), as shown in the examples herein.

[0174] In preferred embodiments, the said diaminobutyric acid acetyltransferase-encoding gene is placed under the control of the strong promoter pPDC1.

[0175] Illustratively, the diaminobutyric acid acetyltransferase gene may be inserted within the LYP1 gene and/or within the MUP3 gene, as it is shown in the examples herein.

[0176] In a particular embodiment, a recombinant yeast according to the invention is such that its genome comprises:

[0177] at least one nucleic acid encoding an homoserine-O-acetyltransferase METX over expressed and/or under the control of an inducible or repressible promoter, and preferably under the control of an inducible or repressible promoter; and

[0178] at least one nucleic acid encoding a diaminobutyric acid acetyltransferase EctA over expressed and/or under the control of an inducible or repressible promoter, and preferably under the control of an inducible or repressible promoter. Ectoine Synthase-Encoding Gene Over Expression and/or Controlled Expression

[0179] In preferred embodiments of a recombinant yeast according to the invention, over expression of an ectoine synthase-encoding gene is obtained by inserting, at selected location(s) of the yeast genome, one or more copies of an expression cassette comprising an ectoine synthase coding sequence. An ectoine synthase and an ectoine synthase-encoding gene that are encompassed by the invention are detailed elsewhere in the present specification.

[0180] In some of these embodiments, the said one or more copies of an expression cassette comprising an ectoine synthase coding sequence comprise(s) regulatory sequences allowing a strong expression of the ectoine synthase, such as a strong promoter that is functional in yeast cells.

[0181] In addition to or as an alternative to these embodiments of a recombinant yeast according to the invention, at least one ectoine synthase-encoding gene can be under the control of an inducible or repressible promoter that is functional in yeast cells.

[0182] Without wishing to be bound by any particular theory, the inventors believe that over expression of an ectoine synthase allows, and consequently increases, the level of conversion of the intermediate metabolite acetyl-2,4-diaminobutyrate into ectoine. The same applies when at least one ectoine synthase coding sequence is under the control of an inducible or repressible promoter.

[0183] In preferred embodiments, the said ectoine synthase-encoding gene is the EctC gene from Halomonas elongata, as shown in the examples herein.

[0184] In preferred embodiments, the said ectoine synthase-encoding gene is placed under the control of the strong promoter pTDH3 and/or the strong promoter pTEF1.

[0185] Illustratively, the ectoine synthase gene may be inserted within the LYP1 gene and/or within the MUP3 gene and/or within the URA3 gene, as it is shown in the examples herein.

Deletion or Under Expression of Homoserine Dehydrogenase

[0186] A recombinant yeast according to the invention is further defined as having a genome in which:

[0187] (i) at least one, preferably all, endogenous nucleic acid encoding an homoserine dehydrogenase HOM6 has been deleted and/or interrupted, and/or

[0188] (ii) at least one, preferably all, nucleic acid encoding an homoserine dehydrogenase HOM6 is independently:

[0189] under the control of an inducible or repressible promoter;

[0190] under the control of a weak promoter; and/or

[0191] in a destabilized form.

[0192] Without wishing to be bound by any particular theory, the inventors believe that an under expression of an homoserine dehydrogenase gene shall increase 2,4-diaminobutyrate production by the recombinant yeast by reducing the consumption of the produced aspartyl-semialdehyde by its conversion into homoserine.

[0193] In some embodiments, under expression of an homoserine dehydrogenase may be rendered conditional, for example by placing the expression of this gene under the control of repressible regulatory sequences, such as inducible or repressible promoters.

[0194] Methods for repressing gene expression, for interrupting target genes or for deleting target genes, are well known from the one skilled in the art.

[0195] Homoserine dehydrogenase under expression also encompasses the insertion of a nucleic acid encoding a destabilized homoserine dehydrogenase. A destabilized homoserine dehydrogenase is a variant of homoserine dehydrogenase that is more rapidly degraded within the yeast cell than the parent homoserine dehydrogenase.

[0196] In preferred embodiments, a destabilized homoserine dehydrogenase consists of a degron-tagged homoserine dehydrogenase protein.

[0197] For example, the homoserine dehydrogenase gene can be interrupted by loxP, or for example by URA3. Kl-loxP, and is thus deleted (which can also be termed inactivated).

[0198] It can alternatively be interrupted by a cassette comprising genes of interest, as illustrated in the examples as filed.

Aspartokinase (HOM3)

[0199] The aspartokinase enzyme is a protein which is described in the art for catalyzing the conversion of L-aspartate in the presence of ATP into 4-phospho-L-aspartate. The aspartokinase encoded by the genome of Saccharomyces cerevisiae may be termed HOM3.

[0200] A method implemented to measure the activity level of aspartokinase belongs to the general knowledge of the one skilled in the art.

[0201] In this regard, the one skilled in the art may advantageously refer to the method described by Stadtman et al. (1961, J Biol Chem, Vol. 236 (7): 2033-2038).

[0202] Preferred aspartokinase in the present specification is an enzyme having an EC number of no EC 2.7.2.4.

[0203] According to a preferred embodiment, the nucleic acid(s) encoding an aspartokinase may be nucleic acid(s) originating from organisms preferably selected in a group comprising prokaryotic organisms and eukaryotic organisms. In some embodiments, the nucleic acid(s) encoding an aspartokinase may be nucleic acid(s) originating from archaebacteria. In some embodiments, the nucleic acid(s) encoding an aspartokinase may be nucleic acid(s) originating from organisms preferably selected from Bacillus subtilis, and yeasts. In some other preferred embodiments, the nucleic acid(s) encoding an aspartokinase may be nucleic acid(s) originating from a yeast, and especially from Saccharomyces cerevisiae.

[0204] According to a yet preferred embodiment, the nucleic acid(s) encoding an aspartokinase may be nucleic acid(s) selected from the group consisting of sequences having at least 25%, advantageously at least 65%, preferably at least 80%, nucleic acid identity with a nucleic acid of SEQ ID NO: 1, and also a biological activity of the same nature. The nucleic acid of SEQ ID NO: 1 encodes an aspartokinase originating from Saccharomyces cerevisiae, that may also be termed HOM3.

[0205] A biological activity of the same nature regarding this sequence is the capacity to code for an enzyme that catalyzes the conversion of L-aspartate in the presence of ATP into 4-phospho-L-aspartate.

[0206] As described herein, a nucleic acid sequence having at least 25% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.

[0207] As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.

[0208] As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.

[0209] For the amino acid sequence of the aspartokinase from Saccharomyces cerevisiae, the one skilled in the art may refer to the accession number NP010972 in the UniProt database, or to SEQ ID NO. 2 described herein.

[0210] According to another particular embodiment, the nucleic acid(s) encoding aspartokinase may be nucleic acid(s) encoding an amino acid sequence selected from the group consisting of sequences having at least 25%, advantageously at least 65%, preferably at least 80%, amino acid identity with the amino acid sequence of SEQ ID NO: 2, and also a biological activity of the same nature. Illustratively, the aspartokinase originating from Aquamarina atlantica has 25% amino acid identity with the aspartokinase of SEQ ID NO. 2.

[0211] A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the conversion of L-aspartate in the presence of ATP into 4-phospho-L-aspartate.

[0212] As described herein, an amino acid sequence having at least 25% amino acid identity with a reference nucleic acid sequence encompasses amino acid sequences having at least 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference nucleic acid sequence.

[0213] As described herein, an amino acid sequence having at least 65% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence.

[0214] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence.

[0215] As above-mentioned, the expression level of the aspartokinase in the present invention is regulated by at least one promoter and at least one terminator, such as herein after defined more in details, which are present in 5' and 3' position respectively of the nucleic acid sequence encoding the said aspartokinase.

[0216] As it is specified elsewhere in the present description, the aspartokinase is overexpressed and/or under the control of an inducible or repressible promoter in a recombinant yeast according to the invention.

[0217] In some embodiments, overexpression of the aspartokinase may result from the control of the corresponding gene by a strong promoter within the said recombinant yeast.

[0218] In some other embodiments, overexpression of the aspartokinase may result from the presence of a plurality of copies of an aspartokinase-encoding sequence within the genome of the said recombinant yeast.

[0219] In still further embodiments, overexpression of aspartokinase may result from both (i) the control of the corresponding gene by a strong promoter within the said recombinant yeast and (ii) the presence of a plurality of copies of an aspartokinase-encoding sequence within the genome the said recombinant yeast.

Aspartate Kinase (AK)

[0220] The aspartate kinase enzyme is a protein which is described in the art for catalyzing the conversion of L-aspartate in the presence of ATP into 4-phospho-L-aspartate. The aspartate kinase encoded by the genome of Bacillus subtilis may be termed AK.

[0221] A method implemented to measure the activity level of aspartate kinase belongs to the general knowledge of the one skilled in the art and is the same as the one indicated previously for aspartokinase.

[0222] According to a preferred embodiment, the nucleic acid(s) encoding an aspartate kinase may be nucleic acid(s) originating from organisms preferably selected in a group comprising prokaryotic organisms and eukaryotic organisms. In some embodiments, the nucleic acid(s) encoding an aspartate kinase may be nucleic acid(s) originating from archaebacteria. In some embodiments, the nucleic acid(s) encoding an aspartate kinase may be nucleic acid(s) originating from organisms preferably selected from Bacillus subtilis, and yeasts. In some other preferred embodiments, the nucleic acid(s) encoding an aspartate kinase may be nucleic acid(s) originating from yeast, and especially from Saccharomyces cerevisiae.

[0223] For the nucleic acid sequence, it may be referred to the one disclosed in the access number NC_000964.3 in the NCBI database.

[0224] According to a yet preferred embodiment, the nucleic acid(s) encoding an aspartate kinase may be nucleic acid(s) selected from the group consisting of sequences having at least 25%, advantageously at least 65%, preferably at least 80%, nucleic acid identity with a nucleic acid of SEQ ID NO. 3, and also a biological activity of the same nature. The nucleic acid of SEQ ID NO. 3 encodes an aspartate kinase originating from Bacillus subtilis, that may also be termed AK.

[0225] A biological activity of the same nature regarding this sequence is the capacity to code for an enzyme that catalyzes the conversion of L-aspartate in the presence of ATP into 4-phospho-L-aspartate.

[0226] As described herein, a nucleic acid sequence having at least 25% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.

[0227] As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.

[0228] As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.

[0229] For the amino acid sequence of the aspartate kinase from Bacillus subtilis, the one skilled in the art may refer to the accession number NP_389558.2 in the UniProt database, or to SEQ ID NO. 4 described herein.

[0230] According to another particular embodiment, the nucleic acid(s) encoding aspartate kinase may be nucleic acid(s) encoding an amino acid sequence selected from the group consisting of sequences having at least 25%, advantageously at least 65%, preferably at least 80%, amino acid identity with the amino acid sequence of SEQ ID NO. 4, and also a biological activity of the same nature. Illustratively, the aspartate kinase originating from Aquamarina atlantica has 25% amino acid identity with the aspartokinase of SEQ ID NO. 4.

[0231] A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the conversion of L-aspartate in the presence of ATP into 4-phospho-L-aspartate.

[0232] As described herein, an amino acid sequence having at least 25% amino acid identity with a reference nucleic acid sequence encompasses amino acid sequences having at least 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.

[0233] As described herein, an amino acid sequence having at least 65% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature.

[0234] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature.

[0235] As above-mentioned, the expression level of the aspartate kinase in the present invention is regulated by at least one promoter and at least one terminator, such as herein after defined more in details, which are present in 5' and 3' position respectively of the nucleic acid sequence encoding the said aspartate kinase.

[0236] As it is specified elsewhere in the present description, the aspartate kinase is overexpressed and/or under the control of an inducible or repressible promoter in a recombinant yeast according to the invention.

[0237] In some embodiments, overexpression of the aspartate kinase may result from the control of the corresponding gene by a strong promoter within the said recombinant yeast.

[0238] In some other embodiments, overexpression of the aspartate kinase may result from the presence of a plurality of copies of an aspartate kinase-encoding sequence within the genome of the said recombinant yeast.

[0239] In still further embodiments, overexpression of aspartate kinase may result from both (i) the control of the corresponding gene by a strong promoter within the said recombinant yeast and (ii) the presence of a plurality of copies of an aspartate kinase-encoding sequence within the genome the said recombinant yeast.

Aspartate-Semialdehyde Dehydrogenase (HOM2)

[0240] The aspartate-semialdehyde dehydrogenase is a protein which is known in the art to catalyze the NADPH-dependent formation of L-aspartate-semialdehyde by the reductive dephosphorylation of L-aspartyl-4-phosphate. The aspartate-semialdehyde dehydrogenase encoded by the genome of Saccharomyces cerevisiae may be termed HOM2.

[0241] A method implemented to measure the activity level of aspartate-semialdehyde dehydrogenase belongs to the general knowledge of the one skilled in the art.

[0242] Preferred aspartate-semialdehyde dehydrogenase in the present specification is an enzyme having an EC number 1.2.1.11.

[0243] According to a preferred embodiment, the nucleic acid(s) encoding an aspartate-semialdehyde dehydrogenase may be nucleic acid(s) originating from organisms preferably selected in a group comprising prokaryotic organisms and eukaryotic organisms. In some embodiments, the nucleic acid(s) encoding an aspartate-semialdehyde dehydrogenase may be nucleic acid(s) originating from archaebacteria. In some preferred embodiments, the nucleic acid(s) encoding an aspartate-semialdehyde dehydrogenase may be nucleic acid(s) originating from yeast, and especially from Saccharomyces cerevisiae.

[0244] According to other preferred embodiment, the nucleic acid encoding an aspartate-semialdehyde dehydrogenase may be a variant or a mutant of the aspartate-semialdehyde dehydrogenase from Saccharomyces cerevisiae, wherein the said variant enzyme or the said mutant enzyme uses both NADH or NADPH for catalyzing reactions. Such variant or mutant enzymes are known in the art and are previously discussed in the present text.

[0245] According to a yet preferred embodiment, the nucleic acid(s) encoding an aspartate-semialdehyde dehydrogenase may be nucleic acid(s) selected from the group consisting of sequences having at least 27%, advantageously at least 65%, preferably at least 80%, nucleic acid identity with a nucleic acid selected in a group consisting of the reference nucleic acid sequences of SEQ ID NO: 5 and SEQ ID NO. 6, and also a biological activity of the same nature. The nucleic acids of SEQ ID NO: 5 and SEQ ID NO. 6 encode an aspartate-semialdehyde dehydrogenase originating from Saccharomyces cerevisiae, that may also be collectively termed HOM2 herein.

[0246] A biological activity of the same nature regarding this sequence is the capacity to code for an enzyme that catalyzes the NADPH-dependent formation of L-aspartate-semialdehyde by the reductive dephosphorylation of L-aspartyl-4-phosphate.

[0247] As described herein, a nucleic acid sequence having at least 27% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%. 88%. 89%. 90%, 91%. 92%. 93%. 94%. 95%. 96%, 97%. 98% and 99% nucleotide identity with the said reference nucleic acid sequences, and also a biological activity of the same nature.

[0248] As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequences, and also a biological activity of the same nature.

[0249] As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.

[0250] For the amino acid sequence of the aspartate-semialdehyde dehydrogenase from Saccharomyces cerevisiae, the one skilled in the art may refer to the accession number NP010442 in the UniProt database, or to SEQ ID NO. 7 described herein.

[0251] According to another particular embodiment, the nucleic acid(s) encoding an aspartate-semialdehyde dehydrogenase may be nucleic acid(s) encoding an amino acid sequence selected from the group consisting of sequences having at least 27%, advantageously at least 65%, preferably at least 80%, amino acid identity with the amino acid sequence of SEQ ID NO: 7, and also a biological activity of the same nature.

[0252] Illustratively, the aspartate-semialdehyde dehydrogenase originating from Lactobacillus wasatchensis has 27% amino acid identity with the aspartate-semialdehyde dehydrogenase of SEQ ID NO. 7.

[0253] A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the NADPH-dependent formation of L-aspartate-semialdehyde by the reductive dephosphorylation of L-aspartyl-4-phosphate.

[0254] As described herein, an amino acid sequence having at least 27% amino acid identity with a reference nucleic acid sequence encompasses amino acid sequences having at least 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.

[0255] As described herein, an amino acid sequence having at least 65% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature.

[0256] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature.

[0257] As above-mentioned, the expression level of the aspartate-semialdehyde dehydrogenase in the present invention is regulated by at least one promoter and at least one terminator, such as herein after defined more in details, which are present in 5' and 3' position respectively of the nucleic acid sequence encoding the said aspartate-semialdehyde dehydrogenase.

[0258] As it is specified elsewhere in the present description, the aspartate-semialdehyde dehydrogenase is overexpressed and/or under the control of an inducible or repressible promoter in a recombinant yeast according to the invention.

[0259] In some embodiments, overexpression of the aspartate-semialdehyde dehydrogenase may result from the control of the corresponding gene by a strong promoter within the said recombinant yeast.

[0260] In some other embodiments, overexpression of the aspartate-semialdehyde dehydrogenase may result from the presence of a plurality of copies of a aspartate-semialdehyde dehydrogenase-encoding sequence within the genome of the said recombinant yeast.

[0261] In still further embodiments, overexpression of the aspartate-semialdehyde dehydrogenase may result from both (i) the control of the corresponding gene by a strong promoter within the said recombinant yeast and (ii) the presence of a plurality of copies of an aspartate-semialdehyde dehydrogenase-encoding sequence within the genome the said recombinant yeast.

Diaminobutyrate Aminotransferase (EctB)

[0262] The diaminobutyrate aminotransferase enzyme is a protein which is described in the art for catalyzing the conversion of aspartyl semialdehyde in the presence of glutamate into 2,4-diaminobutyrate. The diaminobutyrate aminotransferase encoded by the genome of Halomonas elongata may be termed EctB or EctB.He.

[0263] A method implemented to measure the activity level of diaminobutyrate aminotransferase belongs to the general knowledge of the one skilled in the art.

[0264] In this regard, the one skilled in the art may advantageously refer to the method described by Ono H et al., 1999, Journal of Bacteriology, p 91-99.

[0265] Preferred diaminobutyrate aminotransferase in the present specification is an enzyme having an EC number of no 2.6.1.76.

[0266] According to a preferred embodiment, the nucleic acid(s) encoding an diaminobutyrate aminotransferase may be nucleic acid(s) originating from organisms preferably selected in a group comprising prokaryotic organisms and eukaryotic organisms. In some embodiments, the nucleic acid(s) encoding a diaminobutyrate aminotransferase may be nucleic acid(s) originating from archaebacteria. In some embodiments, the nucleic acid(s) encoding a diaminobutyrate aminotransferase may be nucleic acid(s) originating from organisms preferably selected from bacteria, and especially from Pseudomonas aeruginosa, Halomonas elongata or Sporocarcina newyorkensisn, and preferably from Pseudomonas aeruginosa or Halomonas elongata.

[0267] According to a yet preferred embodiment, the nucleic acid(s) encoding a diaminobutyrate aminotransferase may be nucleic acid(s) selected from the group consisting of sequences having at least 35%, advantageously at least 65%, preferably at least 80%, nucleic acid identity with a nucleic acid of SEQ ID NO. 8 or SEQ ID NO. 9, and also a biological activity of the same nature.

[0268] A biological activity of the same nature regarding this sequence is the capacity to code for an enzyme that catalyzes the conversion of aspartyl semialdehyde in the presence of glutamate into 2,4-diaminobutyrate.

[0269] As described herein, a nucleic acid sequence having at least 35% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.

[0270] As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.

[0271] As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.

[0272] For the amino acid sequence of the diaminobutyrate aminotransferase from Halomonas elongata, the one skilled in the art may refer to the accession number WP_013332345.1 in the UniProt database, or to SEQ ID NO. 10 or SEQ ID NO. 11 described herein.

[0273] According to another particular embodiment, the nucleic acid(s) encoding diaminobutyrate aminotransferase may be nucleic acid(s) encoding an amino acid sequence selected from the group consisting of sequences having at least 35%, advantageously at least 65%, preferably at least 80%, amino acid identity with the amino acid sequence of SEQ ID NO. 10 or SEQ ID NO. 11, and also a biological activity of the same nature.

[0274] A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the conversion of aspartyl semialdehyde in the presence of glutamate into 2,4-diaminobutyrate.

[0275] As described herein, an amino acid sequence having at least 35% amino acid identity with a reference nucleic acid sequence encompasses amino acid sequences having at least 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.

[0276] As described herein, an amino acid sequence having at least 65% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature.

[0277] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature.

[0278] As above-mentioned, the expression level of the diaminobutyrate aminotransferase in the present invention is regulated by at least one promoter and at least one terminator, such as herein after defined more in details, which are present in 5' and 3' position respectively of the nucleic acid sequence encoding the said diaminobutyrate aminotransferase.

[0279] As it is specified elsewhere in the present description, the diaminobutyrate aminotransferase is overexpressed and/or under the control of an inducible or repressible promoter in a recombinant yeast according to the invention.

[0280] In some embodiments, overexpression of the diaminobutyrate aminotransferase may result from the control of the corresponding gene by a strong promoter within the said recombinant yeast.

[0281] In some other embodiments, overexpression of the diaminobutyrate aminotransferase may result from the presence of a plurality of copies of a diaminobutyrate aminotransferase-encoding sequence within the genome of the said recombinant yeast.

[0282] In still further embodiments, overexpression of diaminobutyrate aminotransferase may result from both (i) the control of the corresponding gene by a strong promoter within the said recombinant yeast and (ii) the presence of a plurality of copies of a diaminobutyrate aminotransferase-encoding sequence within the genome of the said recombinant yeast.

Homoserine O-Acetyltransferase (MET2; METX)

[0283] The homoserine 0-acetyl transferase enzyme is a protein which is described in the art for catalyzing the reaction between Acetyl-CoA and 2,4-diaminobutyrate into CoA and Acetyl-2,4-diaminobutyrate. The homoserine 0-acetyl transferase encoded by the genome of Saccharomyces cerevisiae may be termed MET2.

[0284] A method implemented to measure the activity level of homoserine 0-acetyltransferase belongs to the general knowledge of the one skilled in the art.

[0285] In this regard, the one skilled in the art may advantageously refer to the method described by Shuzo Yamagata (1987, The Journal of Bacteriology, Vol. 169(8) 3458-3463.

[0286] Preferred homoserine O-acetyltransferase in the present specification is an enzyme having an EC number of no EC 2.3.1.31.

[0287] According to a preferred embodiment, the nucleic acid(s) encoding a homoserine O-acetyltransferase may be nucleic acid(s) originating from organisms preferably selected in a group comprising prokaryotic organisms and eukaryotic organisms. In some embodiments, the nucleic acid(s) encoding a homoserine O-acetyltransferase may be nucleic acid(s) originating from archaebacteria. In some embodiments, the nucleic acid(s) encoding a homoserine O-acetyltransferase may be nucleic acid(s) originating from organisms preferably selected from Corynebacterium glutamicum, and yeasts. In some other preferred embodiments, the nucleic acid(s) encoding a homoserine O-acetyltransferase may be nucleic acid(s) originating from yeast, and especially from Saccharomyces cerevisiae.

[0288] According to a particular embodiment, the nucleic acid encoding an homoserine-O-acetyltransferase METX are nucleic acid from a bacterium, in particular from a bacterium selected, independently, from the group consisting of Corynebacterium glutamicum, Escherichia coli, Haemophilius influenza, Streptomyces lavendulae, Leptospira interrogans, Streptococcus pneumonia and Mycobacterium tuberculosis.

[0289] According to a yet preferred embodiment, the nucleic acid(s) encoding a homoserine O-acetyltransferase may be nucleic acid(s) selected from the group consisting of sequences having at least 27%, advantageously at least 65%, preferably at least 80%, nucleic acid identity with a nucleic acid of SEQ ID NO: 12, and also a biological activity of the same nature. The nucleic acid of SEQ ID NO: 12 encodes a homoserine O-acetyltransferase originating from Saccharomyces cerevisiae, that may also be termed MET2. The homoserine O-acetyltransferase originating from Corynebacterium glutamicum is usually termed METX.

[0290] A biological activity of the same nature regarding this sequence is the capacity to code for an enzyme that catalyzes the reaction between Acetyl-CoA and 2,4-diaminobutyrate into CoA and Acetyl-2,4-diaminobutyrate.

[0291] As described herein, a nucleic acid sequence having at least 27% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.

[0292] As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.

[0293] As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.

[0294] For the amino acid sequence of the homoserine O-acetyltransferase from Saccharomyces cerevisiae, the one skilled in the art may refer to the accession number NP014122 in the UniProt database, or to SEQ ID NO. 13 described herein.

[0295] According to another particular embodiment, the nucleic acid(s) encoding a homoserine O-acetyltransferase may be nucleic acid(s) encoding an amino acid sequence selected from the group consisting of sequences having at least 27%, advantageously at least 65%, preferably at least 80%, amino acid identity with the amino acid sequence of SEQ ID NO: 13, and also a biological activity of the same nature. Illustratively, the homoserine O-acetyltransferase originating from Aquamarina atlantica has 27% amino acid identity with the homoserine O-acetyltransferase of SEQ ID NO. 13.

[0296] A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the reaction between Acetyl-CoA and 2,4-diaminobutyrate into CoA and Acetyl-2,4-diaminobutyrate.

[0297] As described herein, an amino acid sequence having at least 27% amino acid identity with a reference nucleic acid sequence encompasses amino acid sequences having at least 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 5.sup.7%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.

[0298] As described herein, an amino acid sequence having at least 65% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature.

[0299] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature.

[0300] As above-mentioned, the expression level of a homoserine O-acetyltransferase in the present invention is regulated by at least one promoter and at least one terminator, such as herein after defined more in details, which are present in 5' and 3' position respectively of the nucleic acid sequence encoding the said homoserine O-acetyltransferase.

[0301] As it is specified elsewhere in the present description, in some embodiments of the invention, the homoserine O-acetyltransferase is overexpressed and/or under the control of an inducible or repressible promoter in a recombinant yeast according to the invention.

[0302] In some embodiments, overexpression of the homoserine O-acetyltransferase may result from the control of the corresponding gene by a strong promoter within the said recombinant yeast.

[0303] In some other embodiments, overexpression of the homoserine O-acetyltransferase may result from the presence of a plurality of copies of a homoserine O-acetyltransferase-encoding sequence within the genome the said recombinant yeast.

[0304] In still further embodiments, overexpression of the homoserine O-acetyltransferase may result from both (i) the control of the corresponding gene by a strong promoter within the said recombinant yeast and (ii) the presence of a plurality of copies of a homoserine O-acetyltransferase-encoding sequence within the genome the said recombinant yeast.

Diaminobutyric Acid Acetyltransferase (EctA)

[0305] The diaminobutyric acid acetyltransferase enzyme is a protein which is described in the art for catalyzing the conversion of 2,4-diaminobutyrate in the presence of Acetyl-CoA into acetyl-2,4-diaminobutyrate. The diaminobutyric acid acetyltransferase encoded by the genome of Halomonas elongata may be termed EctA or EctA.He.

[0306] A method implemented to measure the activity level of diaminobutyric acid acetyltransferase belongs to the general knowledge of the one skilled in the art.

[0307] In this regard, the one skilled in the art may advantageously refer to the method described by Ono H et al., 1999, Journal of Bacteriology, p 91-99.

[0308] Preferred diaminobutyric acid acetyltransferase in the present specification is an enzyme having an EC number of no 2.3.1.178.

[0309] According to a preferred embodiment, the nucleic acid(s) encoding a diaminobutyric acid acetyltransferase may be nucleic acid(s) originating from organisms preferably selected in a group comprising prokaryotic organisms and eukaryotic organisms. In some embodiments, the nucleic acid(s) encoding a diaminobutyric acid acetyltransferase may be nucleic acid(s) originating from archaebacteria. In some embodiments, the nucleic acid(s) encoding a diaminobutyric acid acetyltransferase may be nucleic acid(s) originating from organisms preferably selected from bacteria, and especially from Chromohalobacter salexigens, Pseudomonas aeruginosa, Thaurea sp.28, or Halomonas elongata.

[0310] According to a yet preferred embodiment, the nucleic acid(s) encoding a diaminobutyric acid acetyltransferase may be nucleic acid(s) selected from the group consisting of sequences having at least 30%, advantageously at least 65%, preferably at least 80%, nucleic acid identity with a nucleic acid of SEQ ID NO. 14, and also a biological activity of the same nature.

[0311] A biological activity of the same nature regarding this sequence is the capacity to code for an enzyme that catalyzes the conversion of 2,4-diaminobutyrate in the presence of Acetyl-CoA into acetyl-2,4-diaminobutyrate.

[0312] As described herein, a nucleic acid sequence having at least 30% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.

[0313] As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.

[0314] As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.

[0315] For the amino acid sequence of the diaminobutyric acid acetyltransferase from Halomonas elongata, the one skilled in the art may refer to the accession number WP_035409657.1 in the UniProt database, or to SEQ ID NO. 15 described herein.

[0316] According to another particular embodiment, the nucleic acid(s) encoding diaminobutyric acid acetyltransferase may be nucleic acid(s) encoding an amino acid sequence selected from the group consisting of sequences having at least 30%, advantageously at least 65%, preferably at least 80%, amino acid identity with the amino acid sequence of SEQ ID NO. 15, and also a biological activity of the same nature.

[0317] A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the conversion of 2,4-diaminobutyrate in the presence of Acetyl-CoA into acetyl-2,4-diaminobutyrate.

[0318] As described herein, an amino acid sequence having at least 30% amino acid identity with a reference nucleic acid sequence encompasses amino acid sequences having at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.

[0319] As described herein, an amino acid sequence having at least 65% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature.

[0320] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature.

[0321] As above-mentioned, the expression level of the diaminobutyric acid acetyltransferase in the present invention is regulated by at least one promoter and at least one terminator, such as herein after defined more in details, which are present in 5' and 3' position respectively of the nucleic acid sequence encoding the said diaminobutyric acid acetyltransferase.

[0322] As it is specified elsewhere in the present description, the diaminobutyric acid acetyltransferase is overexpressed and/or under the control of an inducible or repressible promoter in a recombinant yeast according to the invention.

[0323] In some embodiments, overexpression of the diaminobutyric acid acetyltransferase may result from the control of the corresponding gene by a strong promoter within the said recombinant yeast.

[0324] In some other embodiments, overexpression of the diaminobutyric acid acetyltransferase may result from the presence of a plurality of copies of a diaminobutyric acid acetyltransferase-encoding sequence within the genome of the said recombinant yeast.

[0325] In still further embodiments, overexpression of diaminobutyric acid acetyltransferase may result from both (i) the control of the corresponding gene by a strong promoter within the said recombinant yeast and (ii) the presence of a plurality of copies of a diaminobutyric acid acetyltransferase-encoding sequence within the genome the said recombinant yeast.

Ectoine Synthase (EctC)

[0326] The ectoine synthase enzyme is a protein which is described in the art for catalyzing the conversion of acetyl-2,4-diaminobutyrate into ectoine. The ectoine synthase encoded by the genome of Halomonas elongata may be termed EctC or EctC.He.

[0327] A method implemented to measure the activity level of ectoine synthase belongs to the general knowledge of the one skilled in the art.

[0328] In this regard, the one skilled in the art may advantageously refer to the method described by Ono H et al., 1999, Journal of Bacteriology, p 91-99.

[0329] Preferred ectoine synthase in the present specification is an enzyme having an EC number of no 4.2.1.108.

[0330] According to a preferred embodiment, the nucleic acid(s) encoding an ectoine synthase may be nucleic acid(s) originating from organisms preferably selected in a group comprising prokaryotic organisms and eukaryotic organisms. In some embodiments, the nucleic acid(s) encoding an ectoine synthase may be nucleic acid(s) originating from archaebacteria. In some embodiments, the nucleic acid(s) encoding an ectoine synthase may be nucleic acid(s) originating from organisms preferably selected from bacteria, and especially from Pseudomonas aeruginosa, Halomonas elongata or Micrococcus luteus.

[0331] According to a yet preferred embodiment, the nucleic acid(s) encoding an ectoine synthase may be nucleic acid(s) selected from the group consisting of sequences having at least 35%, advantageously at least 65%, preferably at least 80%, nucleic acid identity with a nucleic acid of SEQ ID NO. 16, and also a biological activity of the same nature.

[0332] A biological activity of the same nature regarding this sequence is the capacity to code for an enzyme that catalyzes the conversion of acetyl-2,4-diaminobutyrate into ectoine.

[0333] As described herein, a nucleic acid sequence having at least 35% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.

[0334] As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.

[0335] As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.

[0336] For the amino acid sequence of the ectoine synthase from Halomonas elongata, the one skilled in the art may refer to the accession number WP_013332346 in the UniProt database, or to SEQ ID NO. 17 described herein.

[0337] According to another particular embodiment, the nucleic acid(s) encoding ectoine synthase may be nucleic acid(s) encoding an amino acid sequence selected from the group consisting of sequences having at least 35%, advantageously at least 65%, preferably at least 80%, amino acid identity with the amino acid sequence of SEQ ID NO. 17, and also a biological activity of the same nature.

[0338] A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the conversion of acetyl-2,4-diaminobutyrate into ectoine.

[0339] As described herein, an amino acid sequence having at least 35% amino acid identity with a reference nucleic acid sequence encompasses amino acid sequences having at least 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.

[0340] As described herein, an amino acid sequence having at least 65% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature.

[0341] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature.

[0342] As above-mentioned, the expression level of the ectoine synthase in the present invention is regulated by at least one promoter and at least one terminator, such as herein after defined more in details, which are present in 5' and 3' position respectively of the nucleic acid sequence encoding the said ectoine synthase.

[0343] As it is specified elsewhere in the present description, the ectoine synthase is overexpressed and/or under the control of an inducible or repressible promoter in a recombinant yeast according to the invention.

[0344] In some embodiments, overexpression of the ectoine synthase may result from the control of the corresponding gene by a strong promoter within the said recombinant yeast.

[0345] In some other embodiments, overexpression of the ectoine synthase may result from the presence of a plurality of copies of an ectoine synthase-encoding sequence within the genome of the said recombinant yeast.

[0346] In still further embodiments, overexpression of ectoine synthase may result from both (i) the control of the corresponding gene by a strong promoter within the said recombinant yeast and (ii) the presence of a plurality of copies of an ectoine synthase-encoding sequence within the genome the said recombinant yeast.

Homoserine Dehydrogenase (HOM6)

[0347] The homoserine dehydrogenase enzyme is a protein which is described in the art for catalyzing the conversion of L-homoserine into L-aspartate 4-semialdehyde, in the presence of NAD or NADP. The homoserine dehydrogenase encoded by the genome of Saccharomyces cerevisiae may be termed HOM6.

[0348] A method implemented to measure the activity level of homoserine dehydrogenase belongs to the general knowledge of the one skilled in the art.

[0349] In this regard, the one skilled in the art may advantageously refer to the method described by Calnyanto et al. (2006, Microbiology, Vol. 152: 105-112).

[0350] Preferred homoserine dehydrogenase in the present specification is an enzyme having an EC number of no 1.1.1.3.

[0351] According to a preferred embodiment, the nucleic acid(s) encoding a homoserine dehydrogenase may be nucleic acid(s) originating from organisms preferably selected in a group comprising prokaryotic organisms and eukaryotic organisms. In some preferred embodiments, the nucleic acid(s) encoding a homoserine dehydrogenase may be nucleic acid(s) originating from a yeast, and especially from Saccharomyces cerevisiae.

[0352] According to a particular embodiment, the nucleic acid(s) encoding a homoserine dehydrogenase may be nucleic acid of SEQ ID NO: 18. The nucleic acid of SEQ ID NO: 18 encodes a homoserine dehydrogenase originating from Saccharomyces, that may also be termed HOM6.

[0353] For the amino acid sequence of the homoserine dehydrogenase from Saccharomyces cerevisiae, the one skilled in the art may refer to the accession number AJR75529 or NP012673 in the UniProt database, or to SEQ ID NO. 19 described herein.

[0354] As above-mentioned, the expression level of the homoserine dehydrogenase in the present invention is regulated by at least one promoter and at least one terminator, such as herein after defined more in details, which are present in 5' and 3' position respectively of the nucleic acid sequence encoding the said homoserine dehydrogenase.

[0355] As it is specified elsewhere in the present description, in some embodiments of the invention, the homoserine dehydrogenase is (a) fully or partially deleted or interrupted, or (b) under the control of an inducible or repressible promoter; under the control of a weak promoter; and/or in a destabilized form, in a recombinant yeast according to the invention.

Specific Embodiments of an Ectoine-Producing Recombinant Yeast

[0356] Aspartate Transaminase Over Expression and/or Controlled Expression

[0357] In preferred embodiments of a recombinant yeast according to the invention, at least one nucleic acid encoding an aspartate transaminase is overexpressed and/or is under the control of an inducible or repressible promoter.

[0358] The aspartate transaminase enzyme (also known as aspartate aminotransferase) is a protein which is described in the art for catalyzing the reaction of L-aspartate and 2-oxoglutarate for producing oxaloacetate and L-glutamate. The aspartate transaminase enzyme encoded by the genome Saccharomyces cerevisiae may be termed AAT2.

[0359] According to these embodiments, over expression of an aspartate transaminase-encoding gene is obtained by inserting, at selected location(s) of the yeast genome, one or more copies of an expression cassette comprising an aspartate transaminase coding sequence. Aspartate transaminase and aspartate-transaminase-encoding gene that are encompassed by the invention are detailed elsewhere in the present specification.

[0360] In some of these embodiments, the said one or more copies of an expression cassette comprising an aspartate transaminase coding sequence comprise regulatory sequences allowing a strong expression of the aspartate transaminase, such as a strong promoter that is functional in yeast cells.

[0361] In addition to or as an alternative to these embodiments of a recombinant yeast according to the invention, at least one aspartate transaminase-encoding gene can be under the control of an inducible or repressible promoter that is functional in yeast cells.

[0362] Without wishing to be bound by any particular theory, the inventors believe that an over expression of an aspartate transaminase may induce a high level of conversion of oxaloacetate into aspartate. The same applies when at least one aspartate transaminase coding sequence is under the control of an inducible or repressible promoter.

[0363] A method implemented to measure the activity level of an aspartate transaminase belongs to the general knowledge of the one skilled in the art.

[0364] In this regard, the one skilled in the art may advantageously refer to the method described in Yagi et al. (1982, Biochem, VOl. 92: 35-43).

[0365] In some embodiments, the said aspartate transaminase-encoding gene is the AAT2 gene from Saccharomyces cerevisiae, as shown in the examples herein.

[0366] In preferred embodiments, the aspartate aminotransferase is encoded by the A. Thaliana AAT2-gene.

[0367] In preferred embodiments, the said aspartate transaminase-encoding gene is placed under the control of the inducible or repressible promoter pACU1 or of the strong promoter pADH1 or of the strong promoter pPGK1.

[0368] Illustratively, the AAT2 gene may be inserted within the CAN1 gene and/or within the GNP1 gene and/or within the MUP3 gene, as it is shown in the examples herein.

[0369] Preferred aspartate transaminase in the present specification is known by the EC number 2.6.1.1.

[0370] The nucleic acid(s) encoding an aspartate transaminase may be nucleic acid(s) originating from organisms preferably selected in a group comprising prokaryotic organisms and eukaryotic organisms. In some embodiments, the nucleic acid(s) encoding an aspartate transaminase may be nucleic acid(s) originating from archaebacteria. In some preferred embodiments, the nucleic acid(s) encoding an aspartate transaminase may be nucleic acid(s) originate(s) from a yeast organism, and most preferably Saccharomyces cerevisiae.

[0371] According to a yet preferred embodiment, the nucleic acid(s) encoding an aspartate transaminase or AAT2 may be nucleic acid(s) selected from the group consisting of sequences having at least 39%, advantageously at least 65%, and preferably at least 80%, nucleic acid identity with the nucleic acid sequences of SEQ ID NO: 20, and also a biological activity of the same nature.

[0372] A biological activity of the same nature regarding this sequence is the capacity to code for an enzyme that catalyzes the reaction of L-aspartate and 2-oxoglutarate for producing oxaloacetate and L-glutamate.

[0373] As described herein, a nucleic acid sequence having at least 39% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the nucleic acid sequence of SEQ ID NO: 20, and also a biological activity of the same nature.

[0374] As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the nucleic acid sequence of SEQ ID NO: 20, and also a biological activity of the same nature.

[0375] As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the nucleic acid sequence of SEQ ID NO: 20, and also a biological activity of the same nature.

[0376] For the amino acid sequence of the aspartate transaminase AAT2 from Saccharomyces cerevisiae, the one skilled in the art may refer to the accession number NP013127 in the UniProt database, or to SEQ ID NO. 21 described herein. Illustratively, the aspartate transaminase originating from E. coli has 39% amino acid identity with the aspartate transaminase AAT2 of SEQ ID NO. 21.

[0377] According to another particular embodiment, the nucleic acid(s) encoding an aspartate transaminase may be nucleic acid(s) encoding an amino acid sequence selected from the group consisting of sequences having at least 39%, advantageously at least 65%, preferably at least 80%, identity with the amino acid sequence of SEQ ID NO: 21, and also a biological activity of the same nature.

[0378] A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the reaction of L-aspartate and 2-oxoglutarate for producing oxaloacetate and L-glutamate.

[0379] As described herein, an amino acid sequence having at least 39% amino acid identity with a reference nucleic acid sequence encompasses amino acid sequences having at least 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the amino acid sequence of SEQ ID NO: 21, and also a biological activity of the same nature.

[0380] As described herein, an amino acid sequence having at least 65% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the amino acid sequence of SEQ ID NO: 21, and also a biological activity of the same nature.

[0381] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the amino acid sequence of SEQ ID NO: 21, and also a biological activity of the same nature.

[0382] As above-mentioned, the expression level of the aspartate transaminase in the present invention is regulated by at least one promoter and at least one terminator, such as herein after defined more in details, which are present in 5' and 3' position respectively of the nucleic acid sequence encoding the aspartate transaminase.

[0383] As it is specified elsewhere in the present description, aspartate transaminase is overexpressed in a recombinant yeast according to the invention.

[0384] In some embodiments, overexpression of aspartate transaminase may result from the control of the corresponding gene by a strong promoter within the said recombinant yeast.

[0385] In some other embodiments, overexpression of aspartate transaminase may result from the presence of a plurality of copies of an aspartate transaminase-encoding sequence within the genome the said recombinant yeast.

[0386] In still further embodiments, overexpression of aspartate transaminase may result from both (i) the control of the corresponding gene by a strong promoter within the said recombinant yeast and (ii) the presence of a plurality of copies of an aspartate transaminase-encoding sequence within the genome the said recombinant yeast.

Glutamate Dehydrogenase Over Expression and/or Controlled Expression

[0387] In preferred embodiments of a recombinant yeast according to the invention, at least one nucleic acid encoding a glutamate dehydrogenase that converts oxo-glutarate to glutamate-encoding gene is overexpressed and/or under the control of an inducible or repressible promoter.

[0388] Accordingly, in a particular embodiment, the genome of a recombinant yeast of the invention is such that at least one nucleic acid encoding a glutamate dehydrogenase that converts oxo-glutarate to glutamate is overexpressed and/or is under the control of an inducible or repressible promoter.

[0389] The glutamate dehydrogenase enzyme (also known as NAD-specific glutamate dehydrogenase) is a protein which is described in the art for catalyzing the transformation of 2-oxoglutarate for producing L-glutamate. Thus, glutamate dehydrogenase is an enzyme specifically involved in the chemical reaction involving the conversion of 2-oxoglutarate to L-glutamate, in the presence of NADH.

[0390] According to these embodiments, over expression of a glutamate dehydrogenase enzyme-encoding gene is obtained by inserting, at selected location(s) of the yeast genome, one or more copies of an expression cassette comprising a glutamate dehydrogenase coding sequence. Glutamate dehydrogenase and a glutamate dehydrogenase-encoding gene that are encompassed by the invention are detailed elsewhere in the present specification.

[0391] In some of these embodiments, the said one or more copies of an expression cassette comprising a glutamate dehydrogenase coding sequence comprise regulatory sequences allowing a strong expression of the glutamate dehydrogenase, such as a strong promoter that is functional in yeast cells.

[0392] In addition to or as an alternative to these embodiments of a recombinant yeast according to the invention, at least one glutamate dehydrogenase-encoding gene can be under the control of an inducible or repressible promoter that is functional in yeast cells.

[0393] Without wishing to be bound by any particular theory the inventors believe that the over expression of the glutamate dehydrogenase, by converting oxoglutarate into glutamate, simultaneously generates NAD. The same applies when at least one glutamate dehydrogenase coding sequence is under the control of an inducible or repressible promoter.

[0394] A method implemented to measure the activity level of glutamate dehydrogenase belongs to the general knowledge of the one skilled in the art.

[0395] In this regard, the one skilled in the art may advantageously refer to the method described in Noor and Punekar (2005, Microbiology, Vol. 151: 1409-1419).

[0396] In preferred embodiments, the said glutamate dehydrogenase-encoding gene encodes for a glutamate dehydrogenase which uses NADH instead of NADPH, is more particularly the GDH gene from Entodinium caudatum (represented as GDH.Eca or GDH2.Eca) or from Saccharomyces cerevisiae (represented as GDH2), as shown in the examples herein. In a particular embodiment, the said glutamate dehydrogenase-encoding gene encodes for a glutamate dehydrogenase from Entodinium caudatum.

[0397] Preferred glutamate dehydrogenase in the present specification can in particular be the enzyme having the EC number no EC 1.4.1.2.

[0398] In preferred embodiments, the said glutamate dehydrogenase-encoding gene is placed under the control of the strong promoter pTDH3 or the inducible or repressible promoter pCUP1-1.

[0399] Illustratively, the glutamate dehydrogenase gene may be inserted within the HIS3 gene and/or within the MUP3 gene, as it is shown in the examples herein.

[0400] According to a preferred embodiment, the nucleic acid(s) encoding a glutamate dehydrogenase may be nucleic acid(s) originating from organisms preferably selected in a group comprising prokaryotic organisms and eukaryotic organisms. In some embodiments, the nucleic acid(s) encoding a glutamate dehydrogenase may be nucleic acid(s) originating from archaebacteria. In some embodiments, the nucleic acid(s) encoding a glutamate dehydrogenase may be nucleic acid(s) originating from organisms preferably selected from Entodinium caudatum, Bacillus subtilis, Clostridium symbiosium.

[0401] According to a yet preferred embodiment, the nucleic acid(s) encoding a glutamate dehydrogenase may be nucleic acid(s) selected from the group consisting of sequences having at least 49%, advantageously at least 65%, preferably at least 80%, nucleic acid identity with the nucleic acid sequences of SEQ ID NO: 22, and also a biological activity of the same nature. The nucleic acid of SEQ ID NO. 22 encodes a glutamate dehydrogenase originating from Entodinium caudatum, the said nucleic acid sequence being codon-optimized for its expression in yeast, and especially in Saccharomyces cerevisiae.

[0402] A biological activity of the same nature regarding this sequence is the capacity to code for an enzyme that catalyzes the transformation of 2-oxoglutarate for producing L-glutamate.

[0403] As described herein, a nucleic acid sequence having at least 49% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the nucleic acid sequence of SEQ ID NO: 22, and also a biological activity of the same nature.

[0404] As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the nucleic acid sequence of SEQ ID NO: 22, and also a biological activity of the same nature.

[0405] As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the nucleic acid sequence of SEQ ID NO: 22, and also a biological activity of the same nature.

[0406] For the amino acid sequence of the glutamate dehydrogenase from Entodinium caudatum, the one skilled in the art may refer to the accession number AAF15393 in the UniProt database, or to SEQ ID NO. 23 described herein. Illustratively, the glutamate dehydrogenase originating from Giardia intestinalis has 49% amino acid identity with the glutamate dehydrogenase of SEQ ID NO. 23.

[0407] According to another particular embodiment, the nucleic acid(s) encoding a glutamate dehydrogenase may be nucleic acid(s) encoding an amino acid sequence selected from the group consisting of sequences having at least 49%, advantageously at least 65%, preferably at least 80%, amino acid identity with the amino acid sequence of SEQ ID NO: 23, and also a biological activity of the same nature.

[0408] A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the transformation of 2-oxoglutarate for producing L-glutamate.

[0409] As described herein, an amino acid sequence having at least 49% amino acid identity with a reference nucleic acid sequence encompasses amino acid sequences having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the amino acid sequence of SEQ ID NO: 23, and also a biological activity of the same nature.

[0410] As described herein, an amino acid sequence having at least 65% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the amino acid sequence of SEQ ID NO: 23, and also a biological activity of the same nature.

[0411] As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the amino acid sequence of SEQ ID NO: 23, and also a biological activity of the same nature.

[0412] As above-mentioned, the expression level of the glutamate dehydrogenase in the present invention is regulated by at least one promoter and at least one terminator, such as herein after defined more in details, which are present in 5' and 3' position respectively of the nucleic acid sequence encoding the said glutamate dehydrogenase.

[0413] As it is specified elsewhere in the present description, the glutamate dehydrogenase is overexpressed in a recombinant yeast according to the invention.

[0414] In some embodiments, overexpression of the glutamate dehydrogenase may result from the control of the corresponding gene by a strong promoter within the said recombinant yeast.

[0415] In some other embodiments, overexpression of the glutamate dehydrogenase may result from the presence of a plurality of copies of a glutamate dehydrogenase-encoding sequence within the genome the said recombinant yeast.

[0416] In still further embodiments, overexpression of the glutamate dehydrogenase may result from both (i) the control of the corresponding gene by a strong promoter within the said recombinant yeast and (ii) the presence of a plurality of copies of a glutamate dehydrogenase-encoding sequence within the genome the said recombinant yeast.

Export of the Compounds of Interest

[0417] In further embodiments of a recombinant yeast according to the invention, the export of the produced ectoine outside of the yeast cell may be enhanced by (i) under expression of genes encoding yeast permeases, by (ii) under expression of genes encoding amino acid exporter proteins, or by (iii) both under expression of genes encoding yeast permeases and under expression of genes encoding amino acid exporter proteins.

Under Expression of Permease-Encoding Gene(s)

[0418] As it is described below, permease-encoding genes that may be under expressed in a recombinant yeast according to the invention encompass AGP1, AGP3, BAP3, BAP2, GAP1, GNP1, MUP3 and MUP1.

[0419] AGP1 is the general amino acid permease 1 from Saccharomyces cerevisiae. For the amino acid sequence of AGP1 it may be referred to the access number NP_009905 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001178671 in the NCBI database.

[0420] AGP3 is the general amino acid permease 3 from Saccharomyces cerevisiae. For the amino acid sequence of AGP3 it may be referred to the access number NP_116600 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001179912 in the NCBI database.

[0421] BAP3 is the valine amino acid permease from Saccharomyces cerevisiae. For the amino acid sequence of BAP3 it may be referred to the access number NP_010331 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001180354 in the NCBI database.

[0422] BAP2 is the Leu/Val/Ile amino acid permease from Saccharomyces cerevisiae. For the amino acid sequence of BAP2 it may be referred to the access number NP_009624 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001178416 in the NCBI database.

[0423] GAP1 is the general amino-acid permease from Saccharomyces cerevisiae.

[0424] For the amino acid sequence of GAP1 it may be referred to the access number NP_012965.3 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001179829 in the NCBI database.

[0425] GNP1 is the high-affinity glutamine permease from Saccharomyces cerevisiae. For the amino acid sequence of GNP1 it may be referred to the access number NP_010796 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001180816 in the NCBI database.

[0426] MUP3 is the low-affinity methionine permease from Saccharomyces cerevisiae. For the amino acid sequence of MUP3 it may be referred to the access number NP_011827 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001179116 in the NCBI database.

[0427] MUP1 is the low-affinity methionine permease from Saccharomyces cerevisiae. For the amino acid sequence of MUP it may be referred to the access number NP_011569 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001181184 in the NCBI database.

[0428] In some embodiments of a recombinant yeast according to the invention, the said recombinant yeast is further defined as having an under expression one or more genes encoding a permease, that encompasses AGP1, AGP3, BAP3, BAP2, GAP1, GNP1, MUP3 and MUP1 permeases.

[0429] Accordingly, in a particular embodiment, the genome of a recombinant yeast of the invention is such that at least one of the following modifications has been performed:

[0430] (A) at least one, preferably all, endogenous nucleic acid encoding a general amino acid permease AGP3 have been deleted from the genome of the yeast, and optionally:

[0431] (i) at least one nucleic acid encoding a general amino acid permease AGP3 has been inserted and is under the control of an inducible or repressible promoter, and/or

[0432] (ii) at least one nucleic acid encoding a destabilized general amino acid permease AGP3 has been inserted;

[0433] (B) at least one, preferably all, endogenous nucleic acid encoding a branched-chain amino-acid permease 3 has been deleted from the genome of the yeast, and, optionally:

[0434] (i) at least one nucleic acid encoding a branched-chain amino-acid permease 3 has been inserted and is under the control of an inducible or repressible promoter, and/or

[0435] (ii) at least one nucleic acid encoding a destabilized branched-chain amino-acid permease 3 has been inserted;

[0436] (C) at least one, preferably all, endogenous nucleic acid encoding a branched-chain amino-acid permease 2 has been deleted from the genome of the yeast, and, optionally:

[0437] (i) at least one nucleic acid encoding a branched-chain amino-acid permease 2 has been inserted and is under the control of an inducible or repressible promoter, and/or

[0438] (ii) at least one nucleic acid encoding a destabilized branched-chain amino-acid permease 2 has been inserted;

[0439] (D) at least one, preferably all, endogenous nucleic acid encoding a general amino acid permease GAP1 has been deleted from the genome of the yeast, and, optionally:

[0440] (i) at least one nucleic acid encoding a general amino acid permease GAP1 has been inserted and is under the control of an inducible or repressible promoter, and/or

[0441] (ii) at least one nucleic acid encoding a destabilized general amino acid permease GAP1 has been inserted;

[0442] (E) at least one, preferably all, endogenous nucleic acid encoding a high-affinity glutamine permease GNP1 has been deleted from the genome of the yeast, and, optionally:

[0443] (i) at least one nucleic acid encoding a high-affinity glutamine permease GNP1 has been inserted and is under the control of an inducible or repressible promoter, and/or

[0444] (ii) at least one nucleic acid encoding a destabilized high-affinity glutamine permease GNP1 has been inserted;

[0445] (F) at least one, preferably all, endogenous nucleic acid encoding a general amino acid permease AGP1 has been deleted from the genome of the yeast, and, optionally:

[0446] (i) at least one nucleic acid encoding a general amino acid permease AGP1 has been inserted and is under the control of an inducible or repressible promoter, and/or

[0447] (ii) at least one nucleic acid encoding a destabilized general amino acid permease AGP1 has been inserted;

[0448] (G) at least one, preferably all, endogenous nucleic acid encoding a low-affinity methionine permease MUP3 has been deleted from the genome of the yeast, and, optionally:

[0449] (i) at least one nucleic acid encoding a low-affinity methionine permease MUP3 has been inserted and is under the control of an inducible or repressible promoter, and/or

[0450] (ii) at least one nucleic acid encoding a destabilized low-affinity methionine permease MUP3 has been inserted;

[0451] (H) at least one, preferably all, endogenous nucleic acid encoding a high-affinity methionine permease MUP1 has been deleted from the genome of the yeast, and, optionally:

[0452] (i) at least one nucleic acid encoding a high-affinity methionine permease MUP1 has been inserted and is under the control of an inducible or repressible promoter, and/or

[0453] (ii) at least one nucleic acid encoding a destabilized high-affinity methionine permease MUP1 has been inserted;

[0454] (I) at least one nucleic acid encoding a probable transporter AQR1 is overexpressed; and/or

[0455] (J) at least one nucleic acid encoding a polyamine transporter 1 is overexpressed.

[0456] In a particular embodiment, at least two, in particular at least three of these modifications have been performed.

[0457] Without wishing to be bound by any particular theory, the inventors believe that an under expression of any of the permease genes shall increase the excretion of the produced ectoine outside the yeast cell, e.g. in the culture medium.

[0458] As regards permeases under expression of one or more of these genes encompasses a complete repression of their expression, e.g. by interruption or deletion of the said one or more permease genes.

[0459] In some embodiments, under expression of a permease-encoding gene may be rendered conditional, for example by placing the expression of this gene under the control of repressible regulatory sequences, such as inducible or repressible promoters.

[0460] Methods for repressing gene expression, for interrupting target genes or for deleting target genes, are well known from the one skilled in the art.

[0461] As regards a permease gene, under expression also encompasses the insertion of a nucleic acid encoding a destabilized permease protein or the insertion of a nucleic acid encoding a destabilized permease protein, or both.

[0462] A destabilized permease is a variant of a permease that is more rapidly degraded within the yeast cell than the parent permease.

[0463] In preferred embodiments, a destabilized permease consists of a degron-tagged permease protein.

[0464] As illustrated in the examples, the AGP3 gene, the BAP3 gene, the GAP1 gene, the GNP1 gene and the MUP3 gene can be interrupted by loxP and are thus deleted.

Over Expression of Amino Acid Exporter Protein-Encoding Gene(s)

[0465] As it is described below, exporter protein-encoding genes that may be over expressed in a recombinant yeast according to the invention encompass AQR1 and TPO1.

[0466] AQR1 is a transporter from Saccharomyces cerevisiae. For the amino acid sequence of AQR1 it may be referred to the access number NP_014334 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001182903 in the NCBI database.

[0467] TPO1 is a polyamine transporter from Saccharomyces cerevisiae. For the amino acid sequence of TPO1 it may be referred to the access number NP_013072 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001181848 in the NCBI database.

[0468] In preferred embodiments of a recombinant yeast according to the invention, over expression of a transporter-encoding gene is obtained by inserting, at selected location(s) of the yeast genome, one or more additional copies of an expression cassette comprising the said transporter coding sequence.

[0469] Without wishing to be bound by any particular theory, the inventors believe that an over expression of a transporter-encoding gene shall increase the excretion of the produced ectoine outside the yeast cell, e.g. in the culture medium.

[0470] In some embodiments, over expression of a transporter-encoding gene is obtained by inserting, at selected location(s) of the yeast genome, one or more additional copies of an expression cassette comprising a transporter gene coding sequence. In some of these embodiments, the said one or more copies of an expression cassette comprising a transporter coding sequence comprise regulatory sequences allowing a strong expression of the said transporter, such as a strong promoter that is functional in yeast cells.

[0471] In some other embodiments, one copy of a transporter-encoding gene is inserted at a selected location of the yeast genome. In these other embodiments, the said one or more copies of an expression cassette comprising a transporter coding sequence comprise regulatory sequences allowing a strong expression of the said transporter, such as a strong promoter that is functional in yeast cells.

[0472] In preferred embodiments, the said amino acid exporter protein-encoding gene AQR1 is placed under the control of the strong promoter pTEF3.

[0473] Illustratively, the AQR1 gene may be inserted within the PYK1 gene, as it is shown in the examples herein.

[0474] In preferred embodiments, the said amino acid exporter protein-encoding gene_TPO1 is placed under the control of the strong inducible or repressible promoter pSAM4 or the strong constitutive promoter pTEF1.

[0475] TPO1-1 can be used instead of TPO1. TPO1-1 is an artificial allele in which the lysines 10, 49, 86, 143, 144 and 145 are replaced by arginines.

[0476] It is believed by the inventors that these modifications protect TPO1 from degradation through the ubiquitin-proteasome pathway, thus stabilizing it.

[0477] In preferred embodiments, the said amino acid exporter protein-encoding gene_TPO1 is placed under the control of the strong promoter pTEF3.

[0478] Illustratively, the TPO1 gene may be inserted within the PYK1 gene, as it is shown in the examples herein.

[0479] In view of further increasing ectoine production, a recombinant yeast according to the invention may comprise additional genetic changes, such that they produce large quantities of the intermediate product oxaloacetate. These optional genetic changes are described here below.

Further Embodiments of an Ectoine-Producing Recombinant Yeast

[0480] According to some embodiments of a recombinant yeast according to the invention, production of ectoine may be further increased by placing the said recombinant yeast in conditions leading to an increase production of the intermediate metabolite oxaloacetate.

[0481] Placing the said recombinant yeast in conditions leading to an increased production of oxaloacetate may be performed by introducing further genetic modifications in the yeast genome.

[0482] The present inventors have found that an optimally increased ectoine production may be reached by introducing further genetic changes to the ectoine-producing recombinant yeast, that are described below.

First Further Embodiments of a Ectoine-Producing Recombinant Yeast

[0483] According to these first further embodiments of a ectoine-producing recombinant yeast according to the invention, further genetic engineering of the recombinant yeast is performed with the aim of increasing the production of the intermediate product phosphoenol-pyruvate (PEP).

[0484] Without wishing to be bound by any particular theory, the inventors believe that the further genetic changes introduced in the ectoine-producing recombinant yeast (i) cause an over-production of NADPH, (ii) cause a controlled and balanced conversion of phosphoenol pyruvate into oxaloacetate and pyruvate, respectively, and (iii) cause a reduced conversion of pyruvate into ethanol and a redirection towards conversion of phosphoenol pyruvate into oxaloacetate.

[0485] These further genetic changes introduced by genetic engineering in a ectoine-producing recombinant yeast according to the invention are specified in more detail below.

[0486] According to these embodiments, genetic changes are introduced so as to over-express a glucose-6-phosphate-1-dehydrogenase (also termed MET19 or ZWF1) and a 6-phosphogluconate dehydrogenase, decarboxylating 1 (also termed GND1). Without wishing to be bound by any particular theory, the inventors believe that an over expression of MET19 and GND1 causes an increase in NADPH production.

[0487] According to these embodiments, genetic changes are introduced so as to over-express a phosphoenolpyruvate carboxylase (also termed PEPC ou PPC) and/or a phosphoenolpyruvate carboxykinase [ATP] (also termed PCK1 or PEPCK).

[0488] According to these embodiments, genetic changes are introduced so as to under-express a pyruvate kinase 1 (also termed PYK1 or CDC19) and a pyruvate kinase 2 (also termed (PYK2). In some of these embodiments, PYK2 gene may be deleted rather than being under-expressed.

[0489] In some of these embodiments, one or more of the genes encoding a pyruvate decarboxylase is (are) inactivated, preferably by deletion. Pyruvate decarboxylase-encoding genes encompass those termed PDC1, PDC5 and PDC6, respectively. According to some of these embodiments, PDC1 and/or PDC6 genes are inactivated, preferably by interruption or deletion, whereas the other pyruvate decarboxylase-encoding gene PDC5 is left unaltered; Or its expression is reduced by controlling it with a weak promoter.

[0490] In some of these embodiments, alcohol dehydrogenase activity of the recombinant yeast is reduced by altering the expression of one or more of the alcohol dehydrogenase-encoding genes. In some of these embodiments, the expression of ADH1 is reduced by placing the gene under the control of a weak promoter or by producing a destabilized ADH1 enzyme. In some of these embodiments, one or more of ADH3, ADH4 and ADH5 may be inactivated, preferably by interruption or deletion.

[0491] In some of these embodiments, an exogenous acetyl dehydrogenase-encoding gene (also termed MHPF) may be introduced in the yeast genome and over-expressed.

[0492] In some of these embodiments, an exogenous acetate kinase-encoding gene (also termed ACKA) may be introduced in the yeast genome and over-expressed.

[0493] In some of these embodiments, an exogenous phosphate acetyl transferase-encoding gene (also termed PTA) may be introduced in the yeast genome and over-expressed.

Glucose-6-phosphate-1-dehydrogenase

[0494] The glucose-6-phosphate-1-dehydrogenase enzyme is a protein which is described in the art for catalyzing D-glucose 6-phosphate to 6-phospho-D-glucono-1,5-lactone, with concomitant reduction of NADP to NADPH.

[0495] A method implemented to measure the activity level of glucose-6-phosphate-1-dehydrogenase belongs to the general knowledge of the one skilled in the art.

[0496] In this regard, the one skilled in the art may advantageously refer to the method described by Kuby, S. et al. (1966) Dehydrogenases and Oxidases Methods in Enzymology 9, 116-117.

[0497] Preferred glucose-6-phosphate-1-dehydrogenase in the present specification is an enzyme having an EC number of no 1.1.1.49.

[0498] For the amino acid sequence of glucose-6-phosphate-1-dehydrogenase (also termed MET19), it may be referred to the access number NP_014158.1 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001183079.1 in the UniProt database.

6-phosphogluconate dehydrogenase, decarboxylating 1

[0499] The 6-phosphogluconate dehydrogenase, decarboxylating 1 enzyme is a protein which is described in the art for catalyzing the oxidative decarboxylation of 6-phosphogluconate to ribulose 5-phosphate and C02, with concomitant reduction of NADP to NADPH.

[0500] A method implemented to measure the activity level of 6-phosphogluconate dehydrogenase, decarboxylating 1 belongs to the general knowledge of the one skilled in the art.

[0501] In this regard, the one skilled in the art may advantageously refer to the method described by He W. et al. (2007) BMC Structural Biology, 7:38.

[0502] Preferred 6-phosphogluconate dehydrogenase, decarboxylating 1 in the present specification is an enzyme having an EC number of no 1.1.1.44.

[0503] For the amino acid sequence of 6-phosphogluconate dehydrogenase, decarboxylating 1 (also termed GND1), it may be referred to the access number NP_012053 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001179314 in the NCBI database.

Pyruvate Kinase 1

[0504] The pyruvate kinase 1 enzyme is a protein which is described in the art for catalyzing the conversion of pyruvate into phosphoenolpyruvate, in the presence of ATP.

[0505] A method implemented to measure the activity level of pyruvate kinase 1 belongs to the general knowledge of the one skilled in the art.

[0506] In this regard, the one skilled in the art may advantageously refer to the method described by Susan-resiga and Nowak (biochemistry, 2004, 43, 15230-15245).

[0507] Preferred pyruvate kinase 1 in the present specification is an enzyme having an EC number of no 2.7.1.40.

[0508] For the amino acid sequence of pyruvate kinase 1 (also termed PYK1) it may be referred to the access number NP_009362 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001178183 in the NCBI database.

[0509] Pyruvate kinase 2 The pyruvate kinase 2 enzyme is a protein which is described in the art for catalyzing the conversion of pyruvate into phosphoenolpyruvate, in the presence of ATP.

[0510] Pyruvate kinase 2 may be used by the yeast cell under conditions in which the level of glycolytic flux is very low.

[0511] A method implemented to measure the activity level of pyruvate kinase 2 belongs to the general knowledge of the one skilled in the art.

[0512] In this regard, the one skilled in the art may advantageously refer to the method described by Susan-resiga and Nowak (biochemistry, 2004, 43, 15230-15245).

[0513] Preferred pyruvate kinase 2 in the present specification is an enzyme having an EC number of no 2.7.1.40.

[0514] For the amino acid sequence of pyruvate kinase 2 (also termed PYK2) it may be referred to the access number NP_014992 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001183767 in the NCBI database.

Pyruvate Decarboxylase Isozyme 1

[0515] The pyruvate decarboxylase isozyme 1 is a protein which is described in the art for being involved in the non-oxidative conversion of pyruvate to acetaldehyde and carbon dioxide during alcoholic fermentation.

[0516] A method implemented to measure the activity level of the pyruvate decarboxylase isozyme 1 belongs to the general knowledge of the one skilled in the art.

[0517] In this regard, the one skilled in the art may advantageously refer to the method described by Wang et al. (Biochemistry, 2001, 40:1755-1763).

[0518] Preferred pyruvate decarboxylase isozyme 1 in the present specification is an enzyme having an EC number of no 4.1.1.1.

[0519] For the amino acid sequence of pyruvate decarboxylase isozyme 1 (also termed PDC1) it may be referred to the access number NP_013145 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001181931 in the NCBI database.

Pyruvate Decarboxylase Isozyme 5

[0520] The pyruvate decarboxylase isozyme 5 is a protein which is described in the art for being involved in the nonoxidative conversion of pyruvate to acetaldehyde and carbon dioxide during alcoholic fermentation.

[0521] A method implemented to measure the activity level of the pyruvate decarboxylase isozyme 5 belongs to the general knowledge of the one skilled in the art.

[0522] In this regard, the one skilled in the art may advantageously refer to the method described by Wang et al. (Biochemistry, 2001, 40:1755-1763).

[0523] Preferred pyruvate decarboxylase isozyme 5 in the present specification is an enzyme having an EC number of no 4.1.1.1.

[0524] For the amino acid sequence of pyruvate decarboxylase isozyme 5 (also termed PDC5) it may be referred to the access number NP_013235 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001182021 in the NCBI database.

Pyruvate Decarboxylase Isozyme 6

[0525] The pyruvate decarboxylase isozyme 6 is a protein which is described in the art for being involved in the nonoxidative conversion of pyruvate to acetaldehyde and carbon dioxide during alcoholic fermentation.

[0526] A method implemented to measure the activity level of the pyruvate decarboxylase isozyme 5 belongs to the general knowledge of the one skilled in the art.

[0527] In this regard, the one skilled in the art may advantageously refer to the method described by Wang et al. (Biochemistry, 2001, 40:1755-1763).

[0528] Preferred pyruvate decarboxylase isozyme 6 in the present specification is an enzyme having an EC number of no 4.1.1.1.

[0529] For the amino acid sequence of pyruvate decarboxylase isozyme 6 (also termed PDC6) it may be referred to the access number NP_013235 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001182021 in the NCBI database.

Acetaldehyde Dehydrogenase

[0530] The acetaldehyde dehydrogenase is a protein which is described in the art for catalyzing the conversion of acetaldehyde to acetyl-CoA, using NAD and coenzyme A.

[0531] A method implemented to measure the activity level of acetaldehyde dehydrogenase belongs to the general knowledge of the one skilled in the art.

[0532] In this regard, the one skilled in the art may advantageously refer to the method described by Fisher et al. (2013) Chemi. Biol. Interact. 202 70-77.

[0533] Preferred acetaldehyde dehydrogenase in the present specification is an enzyme having an EC number of no 1.1.1.10.

[0534] For the amino acid sequence of acetaldehyde dehydrogenase (also termed MHPF) it may be referred to the access number NP_414885 in the UniProt database. For the nucleic acid sequence, it may be referred to the one disclosed in the access number NC 000913.3 in the NCBI database.

Acetate Kinase

[0535] The acetate kinase is a protein which is described in the art for the formation of acetyl phosphate from acetate and ATP.

[0536] A method implemented to measure the activity level of acetate kinase belongs to the general knowledge of the one skilled in the art.

[0537] In this regard, the one skilled in the art may advantageously refer to the method described by Sagers et al. J. Bacteriology (1961) 82 233-238.

[0538] For the amino acid sequence of acetate kinase (also termed ACKA) it may be referred to the access number NP_416799 in the UniProt database. For the nucleic acid sequence, it may be referred to the one disclosed in the access number NC_000913.3 in the NCBI database.

Phosphate Acetyltransferase

[0539] The phosphate acetyltransferase is a protein which is described in the art for catalyzing the reversible interconversion of acetyl-CoA and acetyl phosphate.

[0540] A method implemented to measure the activity level of the phosphate acetyltransferase belongs to the general knowledge of the one skilled in the art.

[0541] In this regard, the one skilled in the art may advantageously refer to the method described by Castano-Cerezo and Canovas, Microbial Cell Factories 2009, 8:54.

[0542] Preferred phosphate acetyltransferase in the present specification is an enzyme having an EC number of no 2.3.1.8.

[0543] For the amino acid sequence of phosphate acetyltransferase (also termed PTA) it may be referred to the access number NP_416800 in the UniProt database. For the nucleic acid sequence, it may be referred to the one disclosed in the access number NC_000913 in the NCBI database.

Alcohol Dehydrogenase 1

[0544] The alcohol dehydrogenase 1 is a protein which is described in the art for catalyzing the conversion of primary unbranched alcohols to their corresponding aldehydes.

[0545] A method implemented to measure the activity level of the alcohol dehydrogenase 1 belongs to the general knowledge of the one skilled in the art.

[0546] In this regard, the one skilled in the art may advantageously refer to the method described by Ganzhorn et al. (1987) The Journal of Biological Chemistry, 262, 3754-61

[0547] Preferred alcohol dehydrogenase 1 in the present specification is an enzyme having an EC number of no 1.1.1.1.

[0548] For the amino acid sequence of alcohol dehydrogenase 1 (also termed ADH1) it may be referred to the access number NP_014555 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001183340 in the NCBI database.

Alcohol Dehydrogenase 3

[0549] The alcohol dehydrogenase 3 is a protein which is described in the art for catalyzing the conversion of primary unbranched alcohols to their corresponding aldehydes.

[0550] A method implemented to measure the activity level of the alcohol dehydrogenase 3 belongs to the general knowledge of the one skilled in the art.

[0551] In this regard, the one skilled in the art may advantageously refer to the method described by Ganzhorn et al. (1987) The Journal of Biological Chemistry, 262, 3754-61.

[0552] Preferred alcohol dehydrogenase 3 in the present specification is an enzyme having an EC number of no 1.1.1.1.

[0553] For the amino acid sequence of alcohol dehydrogenase 3 (also termed ADH3) it may be referred to the access number NP_013800 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001182582 in the NCBI database.

Alcohol Dehydrogenase 4

[0554] The alcohol dehydrogenase 4 is a protein which is described in the art for catalyzing the conversion of primary unbranched alcohols to their corresponding aldehydes.

[0555] A method implemented to measure the activity level of the alcohol dehydrogenase 4 belongs to the general knowledge of the one skilled in the art.

[0556] In this regard, the one skilled in the art may advantageously refer to the method described by Ganzhorn et al. (1987) The Journal of Biological Chemistry, 262, 3754-61.

[0557] Preferred alcohol dehydrogenase 4 in the present specification is an enzyme having an EC number of no 1.1.1.1.

[0558] For the amino acid sequence of alcohol dehydrogenase 4 (also termed ADH4) it may be referred to the access number NP_011258 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001181122 in the NCBI database.

Alcohol Dehydrogenase 5

[0559] The alcohol dehydrogenase 5 is a protein which is described in the art for catalyzing the conversion of primary unbranched alcohols to their corresponding aldehydes.

[0560] A method implemented to measure the activity level of the alcohol dehydrogenase 5 belongs to the general knowledge of the one skilled in the art.

[0561] In this regard, the one skilled in the art may advantageously refer to the method described by Ganzhorn et al. (1987) The Journal of Biological Chemistry, 262, 3754-61.

[0562] Preferred alcohol dehydrogenase 5 in the present specification is an enzyme having an EC number of no 1.1.1.1.

[0563] For the amino acid sequence of alcohol dehydrogenase 5 (also termed ADH5) it may be referred to the access number NP_009703 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001178493 in the NCBI database.

Second Further Embodiments of a Ectoine-Producing Recombinant Yeast

[0564] According to these further embodiments of a ectoine-producing recombinant yeast according to the invention, further genetic engineering of the recombinant yeast is performed with the aim of increasing the production of the intermediate product phosphoenol-pyruvate (PEP).

[0565] Without wishing to be bound by any particular theory, the inventors believe that the further genetic changes introduced in the ectoine-producing recombinant yeast (i) cause an over-production of NADPH, (ii) cause a controlled and balanced conversion of phosphoenol pyruvate into oxaloacetate and pyruvate, respectively, and (iii) cause a reduced conversion of pyruvate into ethanol and a redirection towards conversion of phosphoenol pyruvate into oxaloacetate.

[0566] For this purpose, the inventors have conceived a completely novel metabolic pathway, starting from phosphenolpyruvate and ending with the production of oxaloacetate.

[0567] These further genetic changes introduced by genetic engineering in a ectoine-producing recombinant yeast according to the invention are specified in more detail below.

[0568] According to these embodiments, genetic changes are introduced so as to under express the pyruvate kinase 1 (also termed PYK1), and optionally also pyruvate kinase 2 (also termed PYK2). In some of these embodiments, PYK1 may be under-expressed by placing the gene under the control of a weak promoter or of an inducible or repressible promoter. In some of these embodiments, PYK2 may be inactivated, e.g. by interruption or deletion. In some of these embodiments, PYK1 gene may be deleted rather than being under-expressed. In some of these embodiments, PYK1 gene and PYK2 gene may be deleted rather than being under-expressed.

[0569] According to these embodiments, genetic changes are introduced so as to over-express a phosphoenolpyruvate carboxykinase [ATP] (also termed PCK or PCKA or PEPCK), either (i) by constitutive over-expression or (ii) by inducible over-expression.

[0570] According to these embodiments, genetic changes are introduced so as over-express in the cytoplasm a malate dehydrogenase, such as a peroxisomal malate dehydrogenase (also termed MDH3), either (i) by constitutive over-expression or (ii) by inducible over-expression.

[0571] According to these embodiments, genetic changes are introduced so as over-express a NADP-dependent malic enzyme 3 (also termed ME3 or NADP-ME3), either (i) by constitutive over-expression or (ii) by inducible over-expression.

[0572] According to these embodiments, genetic changes are introduced so as to reduce expression of one or more alcohol dehydrogenase(s), preferably one or more alcohol dehydrogenase(s) selected in a group comprising alcohol dehydrogenase 1 (also termed ADH1), alcohol dehydrogenase 3 (also termed ADH3), alcohol dehydrogenase 4 (also termed ADH4) and alcohol dehydrogenase 5 (also termed ADH5), e.g. (i) by placing the corresponding coding sequence under the control of a weak promoter or of an inducible or repressible promoter, or (ii) by production of a destabilized form of the said alcohol dehydrogenase(s).

[0573] Still according to these embodiments, genetic changes are introduced so as to over-express an exogenous acetaldehyde dehydrogenase (also termed MHPF), either (i) by constitutive over-expression or (ii) by inducible over-expression.

Phosphoenolpyruvate Carboxykinase (PPCK)

[0574] The phosphoenol carboxykinase [ATP] enzyme is a protein which is described in the art for catalyzing the conversion of oxaloacetate to phosphoenolpyruvate through direct phosphoryl transfer between the nucleoside triphosphate and oxaloacetate.

[0575] A method implemented to measure the activity level of phosphoenol carboxykinase [ATP] belongs to the general knowledge of the one skilled in the art.

[0576] In this regard, the one skilled in the art may advantageously refer to the method described by Bazaes S. et al. (2007) The Protein Journal, 26, 265-269 and Marit J. Van der Werf et al. (1997) Arch Microbiol 167:332-342.

[0577] Preferred phosphoenol carboxykinase [ATP] in the present specification is an enzyme having an EC number of no 4.1.1.49.

[0578] For the amino acid sequence of phosphoenol carboxykinase [ATP] (also termed PCKA) it may be referred to the access number NP_417862 in the UniProt database. For the nucleic acid sequence, it may be referred to the one disclosed in the access number NC_000913 in the NCBI database.

[0579] Preferred phosphoenol carboxykinase according to the invention can be selected from phosphoenolpyruvate carboxykinase PPCK such as PEPCK having an EC number of no 4.1.1.32.

Malate Dehydrogenase

[0580] The malate dehydrogenase enzyme is a protein which is described in the art for catalyzing the conversion of malate to oaxaloacetate, in the presence of NADH.

[0581] A method implemented to measure the activity level of malate dehydrogenase belongs to the general knowledge of the one skilled in the art. Mention can for example be made of the commercial kit sold by Sigma entitled "Malate dehydrogenase assay kit" under the reference MAK196-1KT.

[0582] For the amino acid sequence of malate dehydrogenase (also termed MDH3) it may be referred to the access number NP_010205 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_00118037 in the NCBI database.

NADP-Dependent Malic Enzyme 3

[0583] The NADP-dependent malic enzyme 3 enzyme is a protein which is described in the art for catalyzing the conversion of malate to pyruvate, in the presence of NADP.

[0584] A method implemented to measure the activity level of NADP-dependent malic enzyme 3 belongs to the general knowledge of the one skilled in the art.

[0585] In this regard, the one skilled in the art may advantageously refer to the method described by Gerrard-Wheeler et al. FEBS Journal 276 (2009) 5665-5677.

[0586] Preferred NADP-dependent malic enzyme 3 in the present specification is an enzyme having an EC number of no 1.1.1.40.

[0587] For the amino acid sequence of NADP-dependent malic enzyme 3 (also termed NADP-ME3 or ME3) it may be referred to the access number NP_197960 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_122489 in the NCBI database.

[0588] Alcohol dehydrogenase 1, alcohol dehydrogenase 3, alcohol dehydrogenase 4, alcohol dehydrogenase 5 and acetaldehyde dehydrogenase are as indicated here-above.

Promoters

[0589] As it is disclosed herein, the expression of the genes of interest that have been genetically engineered for obtaining a recombinant yeast according to the invention comprise appropriate regulatory sequences that are functional in yeast cells, including in Saccharomyces cerevisiae.

[0590] As disclosed in the present specification, various promoters may be used for the desired expression of the coding sequences of interest, which include (i) constitutive strong promoters (also called strong promoters in the present text), (ii) constitutive weak promoters (also called weak promoters in the present text) and (iii) inducible or repressible promoters. A list of yeast promoter with their relative activities in different media can be found in Keren et al. (2013) Molecular Systems Biology 9:701.

[0591] Promoters allowing the constitutive over-expression of a given gene, may be found in literature (Velculescu et al. (1997) Cell 88, 243-251).

[0592] Strong promoters more particularly interesting in the present invention may be selected from the group comprising:

[0593] pTDH3 (SEQ ID No 24),

[0594] pENO2 (SEQ ID No 25),

[0595] pTEF KI (SEQ ID No 26),

[0596] pTEF3 (SEQ ID No 27),

[0597] pTEF1 (SEQ ID No 28),

[0598] pADH1 (SEQ ID No 29),

[0599] pGMP1 (SEQ ID No 30),

[0600] pFBA1 (SEQ ID No 31),

[0601] pPDC1 (SEQ ID No 32),

[0602] pCCW12 (SEQ ID No 33), and

[0603] pGK1 (SEQ ID No 34).

[0604] According to a particular embodiment, the strong promoter according to the invention is, independently, selected from the group consisting of pTDH3, pENO2, pTEF-KI, pTEF3, pTEF1, pADH1, pGMP1, pFBA1, pPDC1, pCCW12 and pGK1.

[0605] Weak promoters more particularly interesting in the present invention may be selected from the group comprising:

[0606] pURA3 (SEQ ID No 36),

[0607] pRPLA1 (SEQ ID No 37),

[0608] pNUP57 (SEQ ID No 119), and

[0609] pGAP1 (SEQ ID No 120).

[0610] According to a particular embodiment, the weak promoter according to the invention is, independently, selected from the group consisting of pURA3, pRPLA1, pNUP57 andpGAP1.

[0611] As previously mentioned, inducible or repressible promoters are promoters whose activity is controlled by the presence or absence of biotic or abiotic factors and also by the quantity of said factor. Accordingly, for some promoters, their activity will in particular be induced and thus increased when the quantity of a given factor increases or is increased, and, accordingly, the activity of these same promoters can be repressed and thus reduced when the quantity of said factor diminishes or is reduced. The quantity of said factor(s) in the culture medium of a recombinant yeast of the invention comprising inducible or repressible promoters can be decided and thus controlled by the man skilled in the art.

[0612] For example, increasing the quantity of methionine in a culture medium of a recombinant yeast according to the invention comprising a pSAM4 promoter will induce and thus increase transcription of the gene under the control of this promoter. On the contrary, reducing the quantity of methionine in said culture medium will lead to a repression, and thus a reduced, transcription of the gene under the control of this promoter.

[0613] In another example, increasing the quantity of copper in a culture medium of a recombinant yeast according to the invention comprising a pCTR1 promoter will repressed and thus decrease transcription of the gene under the control of this promoter. On the contrary, reducing the quantity of copper in said culture medium will lead to an induced, and thus an increased, transcription of the gene under the control of this promoter.

[0614] For this reason, the following promoters are referred to in the present text as being "inducible or repressible promoters".

[0615] According to a first embodiment, inducible or repressible promoters according to the invention may be selected from the group comprising promoters inducible or repressible with copper, promoters inducible or repressible with methionine and promoters inducible or repressible with threonine, and are in particular selected from the group consisting of:

[0616] pSAM4--methionine inducible or repressible (SEQ ID No 38),

[0617] pCUP1-1--copper inducible or repressible (SEQ ID No 39),

[0618] pCUP1.cgla--copper inducible or repressible (SEQ ID No 40),

[0619] pCUP1.sba--copper inducible or repressible (SEQ ID No 41),

[0620] pACU1--copper inducible or repressible (SEQ ID No 42),

[0621] pACU2--copper inducible or repressible (SEQ ID No 43),

[0622] pACU3p--copper inducible or repressible (SEQ ID No 44),

[0623] pACU4p--copper inducible or repressible (SEQ ID No 45),

[0624] pACU5--copper inducible or repressible (SEQ ID No 46),

[0625] pACU6--copper inducible or repressible (SEQ ID No 47),

[0626] pACU7--copper inducible or repressible (SEQ ID No 48),

[0627] pACU8--copper inducible or repressible (SEQ ID No 49),

[0628] pACU9--copper inducible or repressible (SEQ ID No 50),

[0629] pACU10p--copper inducible or repressible (SEQ ID No 51),

[0630] pACU1 1--copper inducible or repressible (SEQ ID No 52),

[0631] pACU12--copper inducible or repressible (SEQ ID No 53),

[0632] pACU13--copper inducible or repressible (SEQ ID No 54),

[0633] pACU14--copper inducible or repressible (SEQ ID No 55),

[0634] pACU15--copper inducible or repressible (SEQ ID No 56),

[0635] pGAL/CUP1p--copper inducible or repressible (SEQ ID No 57),

[0636] pCRS5--copper inducible or repressible (SEQ ID No 58), and

[0637] pCHA1--threonine inducible or repressible (SEQ ID No 59).

[0638] According to this embodiment, the inducible or repressible promoter according to the invention can in particular, independently, be selected from the group consisting of pSAM4, pCUP1-1, pCUP1.Cgla, pCUP1.Sba, pACU1, pACU2, pACU3p, pACU4p, pACU5, pACU6, pACU7, pACU8, pACU9, pACU10p, pACU11, pACU12, pACU13, pACU14, pACU15, pGAL/CUP1p, pCRS5, andpCHA1.

[0639] The activity of these promoters is thus induced by the increasing presence of methionine, copper or threonine as indicated above, and their activity diminishes, i.e. is repressed, when the quantity of methionine, copper or threonine is reduced.

[0640] According to a second embodiment, inducible or repressible promoters according to the invention may be selected from the group comprising promoters inducible or repressible with copper, promoters inducible or repressible with lysine and promoters inducible or repressible with methionine, and in particular selected from the group consisting of:

[0641] pCTR1--copper inducible or repressible (SEQ ID No 60),

[0642] pCTR3--copper inducible or repressible (SEQ ID No 61),

[0643] pCUR1--copper inducible or repressible (SEQ ID No 62),

[0644] pCUR2--copper inducible or repressible (SEQ ID No 63),

[0645] pCUR3--copper inducible or repressible (SEQ ID No 64),

[0646] pCUR4--copper inducible or repressible (SEQ ID No 65),

[0647] pCUR5p--copper inducible or repressible (SEQ ID No 66),

[0648] pCUR6--copper inducible or repressible (SEQ ID No 67),

[0649] pCUR7--copper inducible or repressible (SEQ ID No 68),

[0650] pCUR8--copper inducible or repressible (SEQ ID No 69),

[0651] pCUR9--copper inducible or repressible (SEQ ID No 70),

[0652] pCUR10--copper inducible or repressible (SEQ ID No 71),

[0653] pCUR11--copper inducible or repressible (SEQ ID No 72),

[0654] pCUR12--copper inducible or repressible (SEQ ID No 73),

[0655] pCUR13--copper inducible or repressible (SEQ ID No 74),

[0656] pCUR14--copper inducible or repressible (SEQ ID No 75),

[0657] pCUR15--copper inducible or repressible (SEQ ID No 76),

[0658] pCUR16--copper inducible or repressible (SEQ ID No 77),

[0659] pCUR17--copper inducible or repressible (SEQ ID No 78),

[0660] pLYS1--lysine inducible or repressible (SEQ ID No 79),

[0661] pLYS4--lysine inducible or repressible (SEQ ID No 80),

[0662] pLYS9--lysine inducible or repressible (SEQ ID No 81),

[0663] pLYR1p--lysine inducible or repressible (SEQ ID No 82),

[0664] pLYR2p--lysine inducible or repressible (SEQ ID No 83),

[0665] pLYR3p--lysine inducible or repressible (SEQ ID No 84),

[0666] pLYR4p--lysine inducible or repressible (SEQ ID No 85),

[0667] pLYR5p--lysine inducible or repressible (SEQ ID No 86),

[0668] pLYR6p--lysine inducible or repressible (SEQ ID No 87),

[0669] pLYR7p--lysine inducible or repressible (SEQ ID No 88),

[0670] pLYR8--lysine inducible or repressible (SEQ ID No 89),

[0671] pLYR9--lysine inducible or repressible (SEQ ID No 90),

[0672] pLYR10--lysine inducible or repressible (SEQ ID No 91),

[0673] pLYR11--lysine inducible or repressible (SEQ ID No 92),

[0674] pMET17--methionine inducible or repressible (SEQ ID No 93),

[0675] pMET6--methionine inducible or repressible (SEQ ID No 94),

[0676] pMET14--methionine inducible or repressible (SEQ ID No 95),

[0677] pMET3--methionine inducible or repressible (SEQ ID No 96),

[0678] pSAM1--methionine inducible or repressible (SEQ ID No 97),

[0679] pSAM2--methionine inducible or repressible (SEQ ID No 98),

[0680] pMDH2--glucose inducible or repressible (SEQ ID No 35),

[0681] pJEN1--glucose inducible or repressible (SEQ ID No 118),

[0682] pICL1--glucose inducible or repressible (SEQ ID No 119),

[0683] pADH2--glucose inducible or repressible (SEQ ID No 120), and

[0684] pMLS1--glucose inducible or repressible (SEQ ID No 121).

[0685] According to this particular embodiment, the inducible or repressible promoter according to the invention can, independently, be selected from the group consisting of pCTR1, pCTR3, pCUR1, pCUR2, pCUR3, pCUR4, pCUR5p, pCUR6, pCUR7, pCUR8, pCUR9, pCUR10, pCUR11, pCUR12, pCUR13, pCUR14, pCUR15, pCUR16, pCUR17, pLYS1, pLYS4, pLYS9, pLYR1p, pLYR2p, pLYR3p, pLYR4p, pLYR5p, pLYR6p, pLYR7p, pLYR8, pLYR9, pLYR10, pLYR11, pMET17, pMET6, pMET14, pMET3, pSAM1, pSAM2, pMDH2, pJEN1, pICL1, pADH2 and pMLS1.

[0686] The activity of these promoters is thus repressed by the increasing presence of methionine, copper, lysine or glucose as indicated above, and their activity increases, i.e. is induced, when the quantity of methionine, copper, lysine or glucose is reduced.

[0687] In a particular embodiment, inducible or repressible promoters according to the invention may be selected from the group comprising promoters inducible or repressible with copper, promoters inducible or repressible with glucose, promoters inducible or repressible with lysine, promoters inducible or repressible with methionine and promoters inducible or repressible with threonine.

[0688] In a more particular embodiment, the inducible or repressible promoter according to the invention can, independently, be selected from the group consisting of pSAM4, pCUP1-1, pCUP1.Cgla, pCUP1.Sba, pACU1, pACU2, pACU3p, pACU4p, pACU5, pACU6, pACU7, pACU8, pACU9, pACU10p, pACU11, pACU12, pACU13, pACU14, pACU15, pGAL/CUP1p, pCRS5, pCHA1, pCTR1, pCTR3, pCUR1, pCUR2, pCUR3, pCUR4, pCUR5p, pCUR6, pCUR7, pCUR8, pCUR9, pCUR10, pCUR11, pCUR12, pCUR13, pCUR14, pCUR15, pCUR16, pCUR17, pLYS1, pLYS4, pLYS9, pLYR1p, pLYR2p, pLYR3p, pLYR4p, pLYR5p, pLYR6p, pLYR7p, pLYR8, pLYR9, pLYR10, pLYR1, pMET17, pMET6, pMET14, pMET3, pSAM1, pSAM2, pMDH2, pJEN1, pICL1, pADH2 and pMLS1.

[0689] More particularly, said promoters, identical or different, may be preferably characterized by a sequence of nucleic acid selected from the group consisting of sequences having at least 80% identity with sequences SEQ ID NO: 24 to 98 and 116-121.

[0690] Synthetic promoters as described in Blazeck & Alper (2013) Biotechnol. J. 8 46-58 can also be used.

[0691] The strong, weak and inductible or repressible promoters of the invention can originate from any organism from the Saccharomycetes class and can in particular originate, independently, from an organism selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces castelii, Saccharomyces bayanus, Saccharomyces arboricola, Saccharomyces kudriavzevii, Ashbya gossypii, Kluveromyces lactis, Pichia pastoris, Candida glabrata, Candida tropicalis, Debaryomyces castelii, Yarrowia lipolitica and Cyberlindnera jadinii.

[0692] The strong, weak and inductible or repressible promoters of the invention can preferably originate from an organism selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces castelii, Saccharomyces bayanus, Saccharomyces arboricola, Saccharomyces kudriavzevii and Kluveromyces lactis.

Terminators

[0693] As it is disclosed herein, the expression of the genes of interest that have been genetically engineered for obtaining a recombinant yeast according to the invention comprise appropriate transcription terminator sequences that are functional in yeast cells, including in Saccharomyces cerevisiae.

[0694] Said transcription terminators, identical or different, may be found in literature Yamanishi et al., (2013) ACS synthetic biology 2, 337-347.

[0695] Terminators more particularly interesting in the present invention may be selected from the group comprising:

[0696] tTDH2 from the gene coding for Glyceraldehyde-3-phosphate dehydrogenase, isozyme 2 (TDH2 gene=Sequence SEQ ID No 99),

[0697] tCYC1 (=Sequence SEQ ID No 100),

[0698] tTDH3 (=Sequence SEQ ID No 101), and

[0699] tADH1 from gene coding for the alcohol dehydrogenase (ADH1 gene=Sequence SEQ ID No 102),

[0700] tADH2 from gene coding for the alcohol dehydrogenase (ADH2 gene=Sequence SEQ ID No 103),

[0701] tTPI1 from the gene encoding for the Triose Phosphate Isomerase (TPIl gene=Sequence SEQ ID No 104),

[0702] tMET17 from the gene encoding for the O-acetyl homoserine-O-acetyl serine sulfhydrylase (Met17 gene=Sequence SEQ ID No 105),

[0703] tENO2 from the gene coding for Enolase II (ENO2 gene=Sequence SEQ ID No 106),

[0704] tMET3 (=Sequence SEQ ID No 107), and

[0705] tPGK1 from the gene encoding for the 3-phosphoglycerate kinase (PGK1 gene=Sequence SEQ ID No 108),

[0706] tDIT1 (=Sequence SEQ ID No 109)

[0707] tRPL3 (=Sequence SEQ ID No 110)

[0708] tRPL41B (=Sequence SEQ ID No 111)

[0709] tRPL15A (=Sequence SEQ ID No 112)

[0710] tIDP1 (=Sequence SEQ ID No 113).

[0711] More particularly, said terminator, identical or different, may be preferably characterized by a sequence of nucleic acid selected from the group consisting of sequences having at least 80% identity with sequences SEQ ID NO: 99 to 113.

Recombinant Yeast

[0712] Generally, yeast can grow rapidly and can be cultivated at higher density as compared with bacteria, and does not require an aseptic environment in the industrial setting. Furthermore, yeast cells can be more easily separated from the culture medium compared to bacterial cells, greatly simplifying the process for product extraction and purification.

[0713] Preferentially, the yeast of the invention may be selected among the genus Saccharomyces, Candida Ashbya, Dekkera, Pichia (Hansenula), Debaryomyces, Clavispora, Lodderomyces, Yarrowia, Zigosaccharomyces, Schizosaccharomyces, Torulaspora, Kluyveromyces, Brettanomycces, Cryptococcus or Malassezia.

[0714] More preferentially, the yeast may be Crabtree positive yeast of genus of Saccharomyces, Dekkera, Schizosaccharomyces, Kluyveromyces, Torulaspora Zigosaccharomyces, or. Brettanomycces.

[0715] More preferentially, the yeast may be from the species Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces douglasii, Saccharomyces bayanus, Zigosaccharomyces bailii, Schizosaccharomyces pombe, Dekkera brucelensis, Dekkera intermedia, Brettanomycces custersii, Brettanomycces intermedius, Kluyveromyces themotolerens, Torulaspora globosa or Torulaspora glabrata.

[0716] More preferentially, the recombinant yeast may belong to the Saccharomyces genus, and preferably to the Saccharomyces cerevisiae species.

[0717] As above-mentioned, a recombinant yeast according to the invention has a pyruvate decarboxylase activity which is reduced by insertion of at least one DNA construct(s) selected from those disclosed in the present specification.

[0718] Methods implemented to insert a specific DNA construct within a gene belong to the general knowledge of a man skilled in the art. A related method is described in more details in the herein after examples.

Culture Conditions

[0719] The present invention also relates to the use of a recombinant yeast such as above-defined, for the production of ectoine.

[0720] The present invention further relates to a method of production of ectoine comprising the following steps:

[0721] providing a recombinant microorganism as previously described, cultivating the recombinant microorganism in a culture medium containing a source of carbon, and

[0722] recovering the ectoine.

[0723] Typically, microorganisms of the invention are grown at a temperature in the range of about 20.degree. C. to about 37.degree. C., preferably at a temperature ranging from 27 to 34.degree. C., in an appropriate culture medium.

[0724] When the recombinant yeast according to the invention belongs to the S. cerevisiae species, the temperature may advantageously range from 27 to 34.degree. C., in an appropriate culture medium.

[0725] Suitable growth media for yeast are common commercially prepared media such as broth that includes yeast nitrogen base, ammonium sulfate, and dextrose as the carbon/energy source) or YPD Medium, a blend of peptone, yeast extract, and dextrose in optimal proportions for growing most. Other defined or synthetic growth media may also be used and the appropriate medium for growth of the particular microorganism will be known by one skilled in the art of microbiology or fermentation science.

[0726] The term "appropriate culture medium" is above-defined.

[0727] Examples of known culture media for a recombinant yeast according to the present invention are known to the person skilled in the art, and are presented in the following publication D. Burke et al., Methods in yeast Genetics--A cold spring harbor laboratory course Manual (2000).

[0728] Suitable pH ranges for the fermentation may be between pH 3.0 to pH 7.5, where pH 4.5 to pH 6.5 is preferred as the initial condition.

[0729] Fermentations may be performed under aerobic conditions or micro-aerobic conditions.

[0730] The amount of product in the fermentation medium can be determined using a number of methods known in the art, for example, high performance liquid chromatography (HPLC) or gas chromatography (GC).

[0731] The present process may employ a batch method of fermentation. A classical batch fermentation is a closed system where the composition of the medium is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation, the medium is inoculated with the desired organism or organisms, and fermentation is permitted to occur without adding anything to the system. Typically, however, a "batch" fermentation is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as temperature, pH and oxygen concentration. In batch systems, the metabolite and biomass compositions of the system change constantly up to the time when the fermentation is stopped. Within batch cultures cells progress through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in log phase generally are responsible for the bulk of production of end product or intermediate.

[0732] A Fed-Batch system may also be used in the present invention. A Fed-Batch system is similar to a typical batch system with the exception that the carbon source substrate is added in increments as the fermentation progresses. Fed-Batch systems are useful when catabolite repression (e.g. glucose repression) is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Measurement of the actual substrate concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases such as CO.sub.2.

[0733] Fermentations are common and well known in the art and examples may be found in Sunderland et al., (1992), herein incorporated by reference. Although the present invention is performed in batch mode it is contemplated that the method would be adaptable to continuous fermentation.

[0734] Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth.

[0735] Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, one method will maintain a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allow all other parameters to vary. In other systems a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions and thus the cell loss due to the medium being drawn off must be balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology.

[0736] It is contemplated that the present invention may be practiced using either batch, fed-batch or continuous processes and that any known mode of fermentation would be suitable. Additionally, it is contemplated that cells may be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for production.

[0737] In order to still improve the ectoine production, a particular embodiment may consist of culturing the recombinant yeast cells in an appropriate culture medium, such as above-mentioned, wherein the said culture medium comprises an optimal amount of carbon source, especially glucose.

[0738] Preferably, the cells are cultured in such an optimal culture medium during only a part of the whole culture duration. In some embodiments, the yeast cells are incubated in the said optimal culture medium 10 hours or more after initiation of the culture, which encompasses 11, 12, 13, 14, 15 or 16 hours or more after initiation of the culture.

[0739] Preferably, the cells are cultured in such an optimal culture medium during a time period ranging from 5 hours to 15 hours, which includes from 6 hours to 10 hours, e.g. 8 hours after initiation of the culture.

[0740] In preferred embodiments, the carbon source comprised in said optimal culture medium consists of glucose. In preferred embodiments, the said optimal culture medium comprises 12% w/w or more glucose, including 15% w/w or more glucose. In preferred embodiments, the said optimal culture medium comprises at most 40% w/w glucose, which includes at most 35% w/w glucose.

[0741] Thus, in the preferred embodiments described above, a method for producing ectoine according to the invention may further comprise, between steps (a) and (c), an intermediate step (b) consisting of cultivating the yeast cells in the said optimal culture medium.

Purification of Ectoine

[0742] According to a specific aspect of the invention, the fermentative production of ectoine comprises a step of isolation of the ectoine from the culture medium. Recovering the ectoine from the culture medium is a routine task for a man skilled in the art. It may be achieved by a number of techniques well known in the art including but not limiting to distillation, gas-stripping, pervaporation, selective precipitation or liquid extraction. The expert in the field knows how to adapt parameters of each technique dependent on the characteristics of the material to be separated.

[0743] The yeast as model of microorganism in the present invention has been retained in that the synthesized ectoine is/are entirely exported outside the cells, thus simplifying the purification process.

[0744] The synthesized ectoine may be collected by distillation. Distillation may involve an optional component different from the culture medium in order to facilitate the isolation of ectoine by forming azeotrope and notably with water. This optional component is an organic solvent such as cyclohexane, pentane, butanol, benzene, toluene, trichloroethylene, octane, diethylether or a mixture thereof.

[0745] Gas stripping is achieved with a stripping gas chosen among helium, argon, carbon dioxide, hydrogen, nitrogen or mixture thereof.

[0746] Liquid extraction is achieved with organic solvent as the hydrophobic phase such as pentane, hexane, heptane or dodecane.

[0747] The terms "between . . . and . . . " and "ranging from . . . to . . . " should be understood as being inclusive of the limits, unless otherwise specified.

[0748] The examples and figures which follow are presented by way of illustration and without implied limitation of the invention.

EXAMPLES

Example 1: Protocol for Making a Recombinant Saccharomyces cerevisiae Strain According to the Invention

[0749] All the hereinafter implemented recombinant Saccharomyces cerevisiae strains were constructed from standard strains using standard yeast molecular genetics procedure (Methods in yeast Genetics--A cold spring harbor laboratory course Manual (2000) by D. Burke, D. Dawson, T. Steams CSHL Press).

[0750] Cluster of the following-mentioned genes were integrated in recombinant yeast at once using the ability of yeast to efficiently recombine free DNA ends which have sequence homology.

[0751] In addition, for a better comprehension of following genotypes:

[0752] ade2, his3, leu2, trp1 and ura3 are auxotrophy marker genes.

[0753] Lowercase letters mean that the considered gene is inactive, uppercase letters reflect an active gene.

[0754] "::": following a gene name means that the gene is interrupted by what follows (if more than one gene are inserted, they are noted in brackets [ ]). The interruption of the gene is concomitant with an entire deletion of the coding sequence but preserves the promoter. In consequence the gene followed by "::" is inactive and is noted in lowercase. If not specified the transcription of the gene inserted is controlled by the promoter of the disrupted gene.

[0755] "gene.Kl" means that the gene originates from Kluyveromyces lactis.

[0756] More particularly, the coding sequences to be cloned were artificially synthetized. For heterologous sequences (non-yeast), the nucleic sequences were modified in order to obtain a synonymous coding sequence using the yeast codon usage. Using restriction enzyme and classical cloning technology, each synthetic sequence was cloned in between a transcription promoter and a transcription terminator. Each promoter sequence is preceded by a 50 to 200 nucleotide sequence homologous to the sequence of the terminator of the upstream gene. Similarly, the terminator of each gene (a gene comprising the promoter-coding sequence-terminator) is followed by sequences homologous to the gene immediately following. So that each of the unit to be integrated have a 50-200 nucleotide overlap with both the unit upstream and the unit downstream. For the first unit, the promoter is preceded by 50-200 nucleotides homologous to the yeast chromosome nucleotide for the locus in which it will be integrated. Similarly, for the last unit, the terminator is followed by 50-200 nucleotides homologous to the yeast chromosome nucleotide for the locus in which it will be integrated.

[0757] Each unit are then PCR amplified from the plasmids constructs, yielding X unit of linear DNA having overlapping sequences. At least one of this gene is an auxotrophic marker, in order to select for recombination event. All the linear fragments are transformed in the yeast at once, and recombinant yeast are selected for the auxotrophy related to the marker used. The integrity of the sequence is then verified by PCR and sequencing.

Example 2: Comparative Examples for the Production of Ectoine

[0758] A. Firstly, two recombinant strains are obtained: YA3370-20 and YA3371-46. These two strains have been recombined in order to only comprise a part of the modifications according to the invention.

[0759] Accordingly, these two strains are as follows:

[0760] YA3370-20: ade2, can1::[pACU1-AAT2-tRPL3-pCUP1-1-PPC-5.Ec-tTPI1]x4, his3::[pACU5-HOM2-2-tRPL3-pTDH3-GDH-2.Eca-tIDP1]x4, hom6::[URA3-pCCW12-ECTB.He-tIDP1]x5, leu2, lypl::[pPDC1-ECTA.He-tCYC1-pTDH3-ECTC.He-tTDH3]x2, pyk1::[LEU2.Kl-RS, pTDH3-PEPCK-1.Ec-tIDP1, pTEF3-AQR1-tRPL41B, pCUR3-PYK1-tPYK1], sam3::[pPDC1-METX.Cg-tRPL3-pTDH3-MHPF.ec-tIDP1]x2, trp1::[pPDC1-PPC-5.Ec-tRPL3-pACU7-AK.Bs-tIDP1-TRP1]x6, ura3

[0761] YA3371-46: ade2, can1::[pACU1-AAT2-tRPL3-pCUP1-1-PPC-5.Ec-tTPI1]x4, his3::[pACU5-HOM2-2-tRPL3-pTDH3-GDH-2.Eca-tIDP1]x4, hom6::[URA3-pCCW12-ECTB.He-tIDP1]x5, leu2, lypl::[pPDC1-ECTA.He-tCYC1-pTDH3-ECTC.He-tTDH3]x2, pyk1::[LEU2.Kl-RS, pTDH3-PEPCK-1.Ec-tIDP1, pTEF3-AQR1-tRPL41B, pCUR3-PYK1-tPYK1], sam3::[pCCW12-ECTB.Pa-tRPL3-pTDH3-MHPF.Ec-tRPL41B]x4, trp1::[pPDC1-PPC-5.Ec-tRPL3-pACU7-AK.Bs-tIDP1-TRP1]x6, ura3

[0762] PEPCK-1 is a form of PEPCK stabilized by modification of the Arginine amino acid in position 2 by a Glycine.

[0763] PPC-5 is a more stable form of PPC wherein an alanine has been added in N+1.

[0764] All these strains were grown for 24 hours in YE (Yeast Extract) 2% and Glucose 8%, and 500 .mu.M of CuSO.sub.4 was added after 8 hours. The content of ectoine in the medium was assayed after 24 hours using the AccQ-Tag precolumn derivatization method for amino acid determination using a AccQ-Tag Ultra Derivatization Kit from Waters as advised by the manufacturer.

[0765] The ectoine amounts obtained with these different strains are respectively:

[0766] YA3370-20: 1 g/L.sup.-1.

[0767] YA3371-46: 1.55 g/L.sup.-1.

[0768] In comparison, a native strain does not produce ectoine.

[0769] It results from this comparative experiment that a recombinant strain comprising the modifications according to the invention produces a greater amount of ectoine when cultured in the same conditions as other recombinant strains not comprising all the genetic modifications according to the invention.

[0770] B. Five other recombinant strains have also been obtained: YA3380-40B, YA3595-25 and YA3595-34.

[0771] These three strains are as follows:

[0772] YA3380-40B: gnp1::[LEU2.Kl-RS, pADH1-AAT2-tRPL15A, pTEF3-MDH3-1-tRPL3, pPDC1-PEPCK-1.Ec-tMET25, pTDH3-MHPF.Ec-tTPI1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tIDP1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tTPI1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tIDP1, pCCW12-ME3.At-tRPL3], his3::[pACU5-ME3.At-tRPL3-pACU6-METX-1.Cg-tIDP1]x11, hom6::[TRPL.Kl, pCCW12.Sba-HOM3-tDIT1], leu2, mup3::[HIS5.Sp, pACU7-PEPCK-1.Ec-tRPL3, pCCW12-HOM2-1-tTDH3, pPGK1-AAT2-tTDH2, pENO2-MDH3-1-tRPL15A, pCUP1-1-GDH-2.Eca-tTPI1, pTDH3.Sba-ECTB.He-tIDP1, pPDC1-ECTA.He-tRPL41B, pTEF1.Sba-ECTC.He-tRPL15A], pyk1::[LEU2.Kl, pTDH3-PEPCK-1.Ec-tIDP1, pPDC1-MDH3-1-tRPL15A, pTEF3-TPO1-tENO2, pCUR3-PYK1-7-tCYC1], trp1, ura3

[0773] YA3595-25: ade2, gnp1::[LEU2.Kl-RS, pADH1-AAT2-tRPL15A, pTEF3-MDH3-1-tRPL3, pPDC1-PEPCK-1.Ec-tMET25, pTDH3-MHPF.Ec-tTPI1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tIDP1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tTPI1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tIDP1, pCCW12-ME3.At-tRPL3], his3, hom6::[TRPL.Kl, pCCW12.Sba-HOM3-tDIT1], leu2, mup3::[HIS5.Sp, pACU7-PEPCK-1.Ec-tRPL3, pCCW12-HOM2-1-tTDH3, pPGK1-AAT2-tTDH2, pENO2-MDH3-1-tRPL15A, pCUP1-1-GDH-2.Eca-tTPI1, pTDH3.Sba-ECTB.He-tIDP1, pPDC1-ECTA.He-tRPL41B, pTEF1.Sba-ECTC.He-tRPL15A], pyk1::[LEU2.Kl, pTDH3-PEPCK-1.Ec-tIDP1, pPDC1-MDH3-1-tRPL15A, pTEF3-TPO1-tENO2, pCUR3-PYK1-7-tCYC1], sam3::[pACU7-PEPCK-1.Ec-tRPL3-pCUP1-1-HOM3-tDIT1]x7, trp1, ura3::[pCCW12-ECTB.Ab-tRPL3-pTDH3-ECTC.He-tRPL41B.Sba]x14

[0774] YA3595-34: ade2, gnp1::[LEU2.Kl-RS, pADH1-AAT2-tRPL15A, pTEF3-MDH3-1-tRPL3, pPDC1-PEPCK-1.Ec-tMET25, pTDH3-MHPF.Ec-tTPI1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tIDP1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tTPI1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tIDP1, pCCW12-ME3.At-tRPL3], his3, hom6::[TRP1.Kl, pCCW12.Sba-HOM3-tDIT1], leu2, mup3::[HIS5.Sp, pACU7-PEPCK-1.Ec-tRPL3, pCCW12-HOM2-1-tTDH3, pPGK1-AAT2-tTDH2, pENO2-MDH3-1-tRPL15A, pCUP1-1-GDH-2.Eca-tTPI1, pTDH3.Sba-ECTB.He-tIDP1, pPDC1-ECTA.He-tRPL41B, pTEF1.Sba-ECTC.He-tRPL15A], pyk1::[LEU2.Kl, pTDH3-PEPCK-1.Ec-tIDP1, pPDC1-MDH3-1-tRPL15A, pTEF3-TPO1-tENO2, pCUR3-PYK1-7-tCYC1], sam3::[pACU7-PEPCK-1.Ec-tRPL3-pCUP1-1-HOM3-tDIT1]x7, trp1, ura3::[pCCW12-ECTB.Ab-tRPL3-pTDH3-ECTC.He-tRPL41B.Sba]x9

[0775] The strains YA3380-40B and YA3595-25 were grown for 24 hours in YPA medium (1% yeast extract, 2% peptone, 0.01% adenine hemisulfate), Glucose 8%, (NH.sub.4).sub.2SO.sub.4 50 mM, and Methionine 0.5 mM and Threonine 0.85 mM. The content of ectoine in the medium was assayed after 24 hours using the AccQ-Tag precolumn derivatization method for amino acid determination using a AccQ-Tag Ultra Derivatization Kit from Waters as advised by the manufacturer.

[0776] PEPCK-1 is a form of PEPCK stabilized by modification of the Arginine amino acid in position 2 by a Glycine.

[0777] The ectoine amounts obtained with these two strains are respectively:

[0778] YA3380-40B: 211 mg/L.sup.-1.

[0779] YA3595-25: 1.29 g/L.sup.-1.

[0780] In comparison, a native strain does not produce ectoine.

[0781] The strain YA3595-34, as well as the strain YA3595-25, were grown for 24 hours in YPA medium (1% yeast extract, 2% peptone, 0.01% adenine hemisulfate), Saccharose 8%, (NH.sub.4).sub.2SO.sub.4 50 mM, and Methionine 0.5 mM and Threonine 0.85 mM. The content of ectoine in the medium was assayed after 24 hours using the AccQ-Tag precolumn derivatization method for amino acid determination using a AccQ-Tag Ultra Derivatization Kit from Waters as advised by the manufacturer.

[0782] The ectoine amounts obtained with these two strains are respectively:

[0783] YA3595-25: 2.63 g/L.sup.-1.

[0784] YA3595-34: 2.58 g/L.sup.-1.

[0785] In comparison, a native strain does not produce ectoine.

[0786] It results from this comparative experiment that a recombinant strain comprising the modifications according to the invention produces a greater amount of ectoine when cultured in the same conditions as other recombinant strains not comprising all the genetic modifications according to the invention.

[0787] C. Three other recombinant strains have also been obtained: YA4440, YA4442 and YA4444.

[0788] These three strains are as follows:

[0789] YA4440: MAT-.alpha., gnp1::[LEU2.Kl-RS, pADH1-AAT2-tRPL15A, pTEF3-MDH3-1-tRPL3, pPDC1-PEPCK-1.Ec-tMET17, pTDH3-MHPF.Ec-tTPI1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tIDP1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tTPI1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tIDP1, pCCW12-ME3.At-tRPL3], his3::[HIS3-pACU5-ME3.At-tRPL3, pACU6-METX-1.Cg-tIDP1]x5, hom6::[TRP1.Kl-RS, pCCW12.Sba-HOM3-tDIT1], leu2, lys2A201, mup3::[HIS5.sp-RS, pACU7-PEPCK-1.Ec-tRPL3, pCCW12-HOM2-1-tTDH3, pPGK1-AAT2-tTDH2, pENO2-MDH3-1-tRPL15A, pCUP1-1-GDH-21.Eca-tTPI1, pTDH3.Sba-ECTB.He-tIDP1, pPDC1-ECTA.He-tRPL41B, pTEF1.Sba-ECTC.He-tRPL15A], pyk1::[LEU2.Kl-RS, pTDH3-PEPCK-1.Ec-tIDP1, pPDC1-MDH3-1-tRPL15A, pTEF3-TPO1-tENO2, pCUR3-PYK1-7-tCYC1], sam3::[pACU7-PEPCK-1.Ec-tRPL3, pCUP1-1-HOM3-tDIT1-sam3]x5, trp1, trp4::[LYS2-loxP, pCCW12-PEPCK-1.Ec-tTPI1, pCCW12-GDH2-tRPL3, pCCW12-METX-1.Cg-tRPL41B.Sba, pCCW12.Sba-HOM3-tRPL15A], ura3::[ECTB.Ab-ECTC.He-URA3]x7

[0790] YA4442: MAT-.alpha., gnp1::[LEU2.Kl-RS, pADH1-AAT2-tRPL15A, pTEF3-MDH3-1-tRPL3, pPDC1-PEPCK-1.Ec-tMET17, pTDH3-MHPF.Ec-tTPI1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tIDP1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tTPI1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tIDP1, pCCW12-ME3.At-tRPL3], his3::[HIS3-pACU5-ME3.At-tRPL3, pACU6-METX-1.Cg-tIDP1]x5, hom6::[TRP1.Kl-RS, pCCW12.Sba-HOM3-tDIT1], leu2, lys2A201, mup3::[HIS5.sp-RS, pACU7-PEPCK-1.Ec-tRPL3, pCCW12-HOM2-1-tTDH3, pPGK1-AAT2-tTDH2, pENO2-MDH3-1-tRPL15A, pCUP1-1-GDH-21.Eca-tTPI1, pTDH3.Sba-ECTB.He-tIDP1, pPDC1-ECTA.He-tRPL41B, pTEF1.Sba-ECTC.He-tRPL15A], pyk1::[LEU2.Kl-RS, pTDH3-PEPCK-1.Ec-tIDP1, pPDC1-MDH3-1-tRPL15A, pTEF3-TPO1-tENO2, pCUR3-PYK1-7-tCYC1], sam3::[pACU7-PEPCK-1.Ec-tRPL3, pCUP1-1-HOM3-tDIT1-sam3]x5, trp1, trp4::[LYS2-loxP, pCCW12-PEPCK-1.Ec-tTPI1, pCCW12-GDH1-tRPL3, pCCW12-METX-1.Cg-tRPL41B.Sba, pCCW12.Sba-HOM3-tRPL15A], ura3::[ECTB.Ab-ECTC.He-URA3]x7

[0791] YA4444: MAT-.alpha., gnp1::[LEU2.Kl-RS, pADH1-AAT2-tRPL15A, pTEF3-MDH3-1-tRPL3, pPDC1-PEPCK-1.Ec-tMET17, pTDH3-MHPF.Ec-tTPI1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tIDP1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tTPI1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tIDP1, pCCW12-ME3.At-tRPL3], his3::[HIS3-pACU5-ME3.At-tRPL3, pACU6-METX-1.Cg-tIDP1]x5, hom6::[TRP1.Kl-RS, pCCW12.Sba-HOM3-tDIT1], leu2, lys2A201, mup3::[HIS5.sp-RS, pACU7-PEPCK-1.Ec-tRPL3, pCCW12-HOM2-1-tTDH3, pPGK1-AAT2-tTDH2, pENO2-MDH3-1-tRPL15A, pCUP1-1-GDH-21.Eca-tTPI1, pTDH3.Sba-ECTB.He-tIDP1, pPDC1-ECTA.He-tRPL41B, pTEF1.Sba-ECTC.He-tRPL15A], pyk1::[LEU2.Kl-RS, pTDH3-PEPCK-1.Ec-tIDP1, pPDC1-MDH3-1-tRPL15A, pTEF3-TPO1-tENO2, pCUR3-PYK1-7-tCYC1], sam3::[pACU7-PEPCK-1.Ec-tRPL3, pCUP1-1-HOM3-tDIT1-sam3]x5, trp1, trp4::[LYS2-loxP, pCCW12-PEPCK-1.Ec-tTPI1, pCCW12-GDH2.Eca-tRPL3, pCCW12-METX-1.Cg-tRPL41B.Sba, pCCW12.Sba-HOM3-tRPL15A], ura3::[ECTB.Ab-ECTC.He-URA3]x7

[0792] GDH1 and GDH2 are endogenous Saccharomyces cerevisiae enzymes, while GDH2.Eca is a GDH enzyme from Entodimium caudatum.

[0793] These strains were grown in Erlenmeyer flasks at 28.degree. C. for 16h in Yeast extract 2%, Sucrose 8%, Methionine 0.5 mM, Threonine 4.2 mM, Urea 50 mM, vitamin B5 4 .mu.M, vitamin B1 6 .mu.M, vitamin B6 10 .mu.M, vitamin B10 1.5 .mu.M, vitamin B3 2.9 .mu.M, vitamin B2 0.5 .mu.M, vitamin B8 0.08 .mu.M, vitamin B9 4.5 nM, CuSO4 500 .mu.M. After 16 hours 500 .mu.M CuSO4 and urea 100 mM were added and the cultures were grown for another 8 hours.

[0794] Ectoine production was then evaluated essentially as described in Ono H, et al. (1999) Journal of Bacteriology, p, 91-99 except that ectoine was detected by HPLC-UV.

[0795] Under these conditions, YA4440 produced 6.4 g/l of ectoine, YA4442 produced 4.7 g/l of ectoine and YA4444 produced 6.1 g/l of ectoine. It is reminded that, in these same conditions, a wild-type strain (e.g. non recombinant) does not a produce a detectable amount of ectoine.

[0796] These three strains are identical but for the GDH enzyme over-expressed. The above results show that the over-expression of NADH dependent GDH (GDH2 in YA4440 and GDH2.Eca in YA4444) allows for the production of more ectoine than the overexpression of a NADPH dependent GDH (GDH1 in YA4442).

[0797] Thus, the overexpression of a NADH dependent glutamate Dehydrogenase allows for the production of more ectoine than the overexpression of a NADPH dependent glutamate Dehydrogenase (GDH1).

Sequence CWU 1

1

12111583DNASaccharomyces cerevisiaeASPARTOKINASE (HOM3)ASPARTOKINASE (HOM3) 1atgccaatgg atttccaacc tacatcaagt cattcgaact gggtcgtgca aaagttcggt 60ggtacatctg tcggtaaatt tcccgtccaa atagtggatg acattgtgaa gcactattct 120aaacctgacg gcccaaacaa taatgtcgct gtcgtttgtt ccgcccgttc ttcatacacc 180aaggctgaag gtaccacttc tcgtcttttg aaatgttgtg atttggcttc gcaagaatct 240gaatttcaag acattatcga agttatcaga caagaccata tcgataatgc cgaccgcttc 300attctcaatc ctgccttgca agccaagtta gtggatgata ccaataaaga acttgaactg 360gtcaagaaat atttaaatgc ttcaaaagtt ttgggtgaag tgagttcacg tacagtagat 420ctggtgatgt catgtggtga gaagttgagt tgtttgttca tgactgcttt atgtaatgac 480cgtggctgta aggccaaata tgtggatttg agccacattg ttccctctga tttcagtgcc 540agcgctttgg ataacagttt ctacactttc ctggttcaag cattgaaaga aaaattggcc 600ccctttgtaa gtgctaaaga gcgtatcgtt ccagtcttta cagggttttt tggtttagtt 660ccaactggtc ttctgaatgg tgttggtcgt ggctataccg atttatgtgc cgctttgata 720gcagttgctg taaatgctga tgaactacaa gtttggaagg aagttgatgg tatatttact 780gctgatcctc gtaaggttcc tgaagcacgt ttgctagaca gtgttactcc agaagaagct 840tctgaattaa catattatgg ttccgaagtt atacatcctt ttacgatgga acaagttatt 900agggctaaga ttcctattag aatcaagaat gttcaaaatc cattaggtaa cggtaccatt 960atctacccag ataatgtagc aaagaagggt gaatctactc caccacatcc tcctgagaac 1020ttatcctcat ctttctatga aaagagaaag agaggtgcca ctgctatcac caccaaaaat 1080gacattttcg tcatcaacat tcattccaat aagaaaaccc tatcccatgg tttcctagct 1140caaatattta ccatcctgga taagtacaag ttagtcgtag atttaatatc tacttctgaa 1200gttcatgttt cgatggcttt gcccattcca gatgcagact cattaaaatc tctgagacaa 1260gctgaggaaa aattgagaat tttaggttct gttgatatca caaagaagtt gtctattgtt 1320tcattagttg gtaaacatat gaaacaatac atcggcattg ctggtaccat gtttactact 1380cttgctgaag aaggcatcaa cattgaaatg atttctcaag gggcaaatga aataaacata 1440tcctgcgtta tcaatgaatc tgactccata aaagcgctac aatgtattca tgccaagtta 1500ctaagtgagc ggacaaatac ttcaaaccaa tttgaacatg ccattgatga acgtttagaa 1560caattgaaaa gacttggaat taa 15832526PRTSaccharomyces cerevisiaeASPARTOKINASE (HOM3)ASPARTOKINASE (HOM3) 2Pro Met Asp Phe Gln Pro Thr Ser Ser His Ser Asn Trp Val Val Gln1 5 10 15Lys Phe Gly Gly Thr Ser Val Gly Lys Phe Pro Val Gln Ile Val Asp 20 25 30Asp Ile Val Lys His Tyr Ser Lys Pro Asp Gly Pro Asn Asn Asn Val 35 40 45Ala Val Val Cys Ser Ala Arg Ser Ser Tyr Thr Lys Ala Glu Gly Thr 50 55 60Thr Ser Arg Leu Leu Lys Cys Cys Asp Leu Ala Ser Gln Glu Ser Glu65 70 75 80Phe Gln Asp Ile Ile Glu Val Ile Arg Gln Asp His Ile Asp Asn Ala 85 90 95Asp Arg Phe Ile Leu Asn Pro Ala Leu Gln Ala Lys Leu Val Asp Asp 100 105 110Thr Asn Lys Glu Leu Glu Leu Val Lys Lys Tyr Leu Asn Ala Ser Lys 115 120 125Val Leu Gly Glu Val Ser Ser Arg Thr Val Asp Leu Val Met Ser Cys 130 135 140Gly Glu Lys Leu Ser Cys Leu Phe Met Thr Ala Leu Cys Asn Asp Arg145 150 155 160Gly Cys Lys Ala Lys Tyr Val Asp Leu Ser His Ile Val Pro Ser Asp 165 170 175Phe Ser Ala Ser Ala Leu Asp Asn Ser Phe Tyr Thr Phe Leu Val Gln 180 185 190Ala Leu Lys Glu Lys Leu Ala Pro Phe Val Ser Ala Lys Glu Arg Ile 195 200 205Val Pro Val Phe Thr Gly Phe Phe Gly Leu Val Pro Thr Gly Leu Leu 210 215 220Asn Gly Val Gly Arg Gly Tyr Thr Asp Leu Cys Ala Ala Leu Ile Ala225 230 235 240Val Ala Val Asn Ala Asp Glu Leu Gln Val Trp Lys Glu Val Asp Gly 245 250 255Ile Phe Thr Ala Asp Pro Arg Lys Val Pro Glu Ala Arg Leu Leu Asp 260 265 270Ser Val Thr Pro Glu Glu Ala Ser Glu Leu Thr Tyr Tyr Gly Ser Glu 275 280 285Val Ile His Pro Phe Thr Met Glu Gln Val Ile Arg Ala Lys Ile Pro 290 295 300Ile Arg Ile Lys Asn Val Gln Asn Pro Leu Gly Asn Gly Thr Ile Ile305 310 315 320Tyr Pro Asp Asn Val Ala Lys Lys Gly Glu Ser Thr Pro Pro His Pro 325 330 335Pro Glu Asn Leu Ser Ser Ser Phe Tyr Glu Lys Arg Lys Arg Gly Ala 340 345 350Thr Ala Ile Thr Thr Lys Asn Asp Ile Phe Val Ile Asn Ile His Ser 355 360 365Asn Lys Lys Thr Leu Ser His Gly Phe Leu Ala Gln Ile Phe Thr Ile 370 375 380Leu Asp Lys Tyr Lys Leu Val Val Asp Leu Ile Ser Thr Ser Glu Val385 390 395 400His Val Ser Met Ala Leu Pro Ile Pro Asp Ala Asp Ser Leu Lys Ser 405 410 415Leu Arg Gln Ala Glu Glu Lys Leu Arg Ile Leu Gly Ser Val Asp Ile 420 425 430Thr Lys Lys Leu Ser Ile Val Ser Leu Val Gly Lys His Met Lys Gln 435 440 445Tyr Ile Gly Ile Ala Gly Thr Met Phe Thr Thr Leu Ala Glu Glu Gly 450 455 460Ile Asn Ile Glu Met Ile Ser Gln Gly Ala Asn Glu Ile Asn Ile Ser465 470 475 480Cys Val Ile Asn Glu Ser Asp Ser Ile Lys Ala Leu Gln Cys Ile His 485 490 495Ala Lys Leu Leu Ser Glu Arg Thr Asn Thr Ser Asn Gln Phe Glu His 500 505 510Ala Ile Asp Glu Arg Leu Glu Gln Leu Lys Arg Leu Gly Ile 515 520 52531215DNABacillus subtilisASPARTATE KINASE (AK)ASPARTATE KINASE (AK) 3atggctatta tcgtccaaaa attcggagga actagcgtta aggatgacaa agggagaaag 60ttggccttag ggcacattaa ggaggcaatt tcagagggtt ataaggtggt tgtagttgta 120tcggctatgg gtagaaaagg ggacccctac gcgacggact cactattggg tttactttac 180ggggatcaat cagcaatcag cccaagagag caggatctgc tgctatcatg tggagaaacc 240atatcctcgg ttgtgttcac cagcatgtta ttagataatg gagtaaaagc agcagccctg 300acgggagccc aggctggttt tttaaccaac gatcagcata ctaatgcaaa aattatagag 360atgaagcctg aacgtctttt cagtgttctt gcaaaccacg acgcagttgt cgtcgctgga 420tttcagggcg ctaccgagaa aggagatact accacaatcg gtagaggtgg ctcggacacg 480tcagctgcag ccctaggtgc tgctgttgat gcagagtaca tagatatctt tactgacgta 540gaaggggtga tgaccgcaga tccaagagta gtagaaaatg caaagccact accagtggta 600acttataccg aaatctgcaa cttggcttac caaggtgcta aggtaatatc tccaagagct 660gtggaaattg ctatgcaagc aaaggttcct atccgtgtta ggagtactta ttcaaacgat 720aaaggtacgt tagtaactag tcatcatagt tccaaagttg gctctgacgt ctttgaaagg 780ttaatcactg gtatcgcaca tgttaaagac gtcactcaat tcaaggtccc ggcgaaaata 840ggtcaatata acgttcaaac agaagtgttt aaagcgatgg cgaatgccgg tatatctgtc 900gatttcttta atattacacc ctctgaaata gtatatacag tcgcgggtaa taagactgaa 960acagctcaaa ggattttgat ggatatgggc tatgatccta tggtcacaag aaattgtgcc 1020aaggtgtctg ccgtgggtgc tggcattatg ggtgtcccag gtgtgacatc gaaaattgtt 1080tctgccttat ctgaaaaaga aattccgatt ttgcaatctg ctgattccca tacaacaatt 1140tgggttttgg ttcatgaagc cgatatggtt cctgctgtta atgccttgca cgaagttttt 1200gaattgtcca aataa 12154404PRTBacillus subtilisASPARTATE KINASE (AK)ASPARTATE KINASE (AK) 4Met Ala Ile Ile Val Gln Lys Phe Gly Gly Thr Ser Val Lys Asp Asp1 5 10 15Lys Gly Arg Lys Leu Ala Leu Gly His Ile Lys Glu Ala Ile Ser Glu 20 25 30Gly Tyr Lys Val Val Val Val Val Ser Ala Met Gly Arg Lys Gly Asp 35 40 45Pro Tyr Ala Thr Asp Ser Leu Leu Gly Leu Leu Tyr Gly Asp Gln Ser 50 55 60Ala Ile Ser Pro Arg Glu Gln Asp Leu Leu Leu Ser Cys Gly Glu Thr65 70 75 80Ile Ser Ser Val Val Phe Thr Ser Met Leu Leu Asp Asn Gly Val Lys 85 90 95Ala Ala Ala Leu Thr Gly Ala Gln Ala Gly Phe Leu Thr Asn Asp Gln 100 105 110His Thr Asn Ala Lys Ile Ile Glu Met Lys Pro Glu Arg Leu Phe Ser 115 120 125Val Leu Ala Asn His Asp Ala Val Val Val Ala Gly Phe Gln Gly Ala 130 135 140Thr Glu Lys Gly Asp Thr Thr Thr Ile Gly Arg Gly Gly Ser Asp Thr145 150 155 160Ser Ala Ala Ala Leu Gly Ala Ala Val Asp Ala Glu Tyr Ile Asp Ile 165 170 175Phe Thr Asp Val Glu Gly Val Met Thr Ala Asp Pro Arg Val Val Glu 180 185 190Asn Ala Lys Pro Leu Pro Val Val Thr Tyr Thr Glu Ile Cys Asn Leu 195 200 205Ala Tyr Gln Gly Ala Lys Val Ile Ser Pro Arg Ala Val Glu Ile Ala 210 215 220Met Gln Ala Lys Val Pro Ile Arg Val Arg Ser Thr Tyr Ser Asn Asp225 230 235 240Lys Gly Thr Leu Val Thr Ser His His Ser Ser Lys Val Gly Ser Asp 245 250 255Val Phe Glu Arg Leu Ile Thr Gly Ile Ala His Val Lys Asp Val Thr 260 265 270Gln Phe Lys Val Pro Ala Lys Ile Gly Gln Tyr Asn Val Gln Thr Glu 275 280 285Val Phe Lys Ala Met Ala Asn Ala Gly Ile Ser Val Asp Phe Phe Asn 290 295 300Ile Thr Pro Ser Glu Ile Val Tyr Thr Val Ala Gly Asn Lys Thr Glu305 310 315 320Thr Ala Gln Arg Ile Leu Met Asp Met Gly Tyr Asp Pro Met Val Thr 325 330 335Arg Asn Cys Ala Lys Val Ser Ala Val Gly Ala Gly Ile Met Gly Val 340 345 350Pro Gly Val Thr Ser Lys Ile Val Ser Ala Leu Ser Glu Lys Glu Ile 355 360 365Pro Ile Leu Gln Ser Ala Asp Ser His Thr Thr Ile Trp Val Leu Val 370 375 380His Glu Ala Asp Met Val Pro Ala Val Asn Ala Leu His Glu Val Phe385 390 395 400Glu Leu Ser Lys51098DNASaccharomyces cerevisiaeASPARTATE-SEMIALDEHYDE DEHYDROGENASE (HOM2)ASPARTATE-SEMIALDEHYDE DEHYDROGENASE (HOM2) 5atggctggaa agaaaattgc tggtgttttg ggtgctactg gttccgttgg tcaacgtttc 60attctgttgt tggcaaatca ccctcatttc gaactgaaag ttcttggtgc ctcttctaga 120tcagctggca agaaatacgt tgacgctgtg aactggaagc aaaccgattt gctaccggaa 180tctgctaccg atattattgt ttccgaatgt aaatctgaat tctttaaaga gtgtgacatc 240gtcttttccg gattggatgc tgactatgct ggcgctatcg aaaaggaatt catggaagct 300ggtatcgcca ttgtttccaa tgccaagaat tatagaagag aacaagatgt gccattgatt 360gttcctgttg tcaatcctga gcatttggat attgtagctc aaaagcttga caccgccaag 420gctcaaggta agccaagacc agggttcatt atctgtattt ccaattgttc cactgcaggt 480ttggttgcac cattgaagcc tttgattgaa aaattcggtc ctattgatgc tttgaccact 540actactttgc aagcaatctc aggtgctggt ttctccccag gtgtaccagg tattgatatt 600ctagacaata ttattccata cattggtggt gaagaagaca agatggaatg ggagaccaag 660aaaatcttgg ctccattagc agaagacaag acacacgtca aactattgac tccagaagaa 720atcaaagtct ctgctcaatg taacagagtc gctgtttccg atgggcacac cgaatgtatc 780tctttgaggt tcaagaacag acctgctcca tccgtcgagc aagtcaagac atgcctaaaa 840gaatacgtct gcgatgccta caaattaggc tgtcattctg ctccaaagca aactattcat 900gttttggaac aaccagacag acctcaacca aggttggaca ggaacagaga cagcggttac 960ggtgtttccg ttggtagaat cagagaagac ccattgttag atttcaaaat ggttgtcctt 1020tcccacaaca ccattattgg tgccgctggt tctggtgtct tgattgccga aatcttacta 1080gcaagaaact tgatttaa 109861098DNASaccharomyces cerevisiaeASPARTATE-SEMIALDEHYDE DEHYDROGENASE (HOM2)ASPARTATE-SEMIALDEHYDE DEHYDROGENASE (HOM2) 6atggctggaa agaaaattgc tggtgttttg ggtgctactg gttccgttgg tcaacgtttc 60attctgttgt tggcaaatca ccctcatttc gaactgaaag ttcttggtgc ctctgagaga 120tcagctggca agaaatacgt tgacgctgtg aactggaagc aaaccgattt gctaccggaa 180tctgctaccg atattattgt ttccgaatgt aaatctgaat tctttaaaga gtgtgacatc 240gtcttttccg gattggatgc tgactatgct ggcgctatcg aaaaggaatt catggaagct 300ggtatcgcca ttgtttccaa tgccaagaat tatagaagag aacaagatgt gccattgatt 360gttcctgttg tcaatcctga gcatttggat attgtagctc aaaagcttga caccgccaag 420gctcaaggta agccaagacc agggttcatt atctgtattt ccaattgttc cactgcaggt 480ttggttgcac cattgaagcc tttgattgaa aaattcggtc ctattgatgc tttgaccact 540actactttgc aagcaatctc aggtgctggt ttctccccag gtgtaccagg tattgatatc 600ctagacaata ttattccata cattggtggt gaagaagaca agatggaatg ggagaccaag 660aaaatcttgg ctccattagc agaagacaag acacacgtca aactattgac tccagaagaa 720atcaaagtct ctgctcaatg taacagagtc gctgtttccg atgggcacac cgaatgtatc 780tctttgaggt tcaagaacag acctgctcca tccgtcgagc aagtcaagac atgcctaaaa 840gaatacgtct gcgatgccta caaattaggc tgtcattctg ctccaaagca aactattcat 900gttttggaac aaccagacag acctcaacca aggttggaca ggaacagaga cagcggttac 960ggtgtttccg ttggtagaat cagagaagac ccattgttag atttcaaaat ggttgtcctt 1020tcccacaaca ccattattgg tgccgctggt tctggtgtct tgattgccga aatcttacta 1080gcaagaaact tgatttaa 10987365PRTSaccharomyces cerevisiaeASPARTATE-SEMIALDEHYDE DEHYDROGENASE (HOM2)ASPARTATE-SEMIALDEHYDE DEHYDROGENASE (HOM2) 7Met Ala Gly Lys Lys Ile Ala Gly Val Leu Gly Ala Thr Gly Ser Val1 5 10 15Gly Gln Arg Phe Ile Leu Leu Leu Ala Asn His Pro His Phe Glu Leu 20 25 30Lys Val Leu Gly Ala Ser Ser Arg Ser Ala Gly Lys Lys Tyr Val Asp 35 40 45Ala Val Asn Trp Lys Gln Thr Asp Leu Leu Pro Glu Ser Ala Thr Asp 50 55 60Ile Ile Val Ser Glu Cys Lys Ser Glu Phe Phe Lys Glu Cys Asp Ile65 70 75 80Val Phe Ser Gly Leu Asp Ala Asp Tyr Ala Gly Ala Ile Glu Lys Glu 85 90 95Phe Met Glu Ala Gly Ile Ala Ile Val Ser Asn Ala Lys Asn Tyr Arg 100 105 110Arg Glu Gln Asp Val Pro Leu Ile Val Pro Val Val Asn Pro Glu His 115 120 125Leu Asp Ile Val Ala Gln Lys Leu Asp Thr Ala Lys Ala Gln Gly Lys 130 135 140Pro Arg Pro Gly Phe Ile Ile Cys Ile Ser Asn Cys Ser Thr Ala Gly145 150 155 160Leu Val Ala Pro Leu Lys Pro Leu Ile Glu Lys Phe Gly Pro Ile Asp 165 170 175Ala Leu Thr Thr Thr Thr Leu Gln Ala Ile Ser Gly Ala Gly Phe Ser 180 185 190Pro Gly Val Pro Gly Ile Asp Ile Leu Asp Asn Ile Ile Pro Tyr Ile 195 200 205Gly Gly Glu Glu Asp Lys Met Glu Trp Glu Thr Lys Lys Ile Leu Ala 210 215 220Pro Leu Ala Glu Asp Lys Thr His Val Lys Leu Leu Thr Pro Glu Glu225 230 235 240Ile Lys Val Ser Ala Gln Cys Asn Arg Val Ala Val Ser Asp Gly His 245 250 255Thr Glu Cys Ile Ser Leu Arg Phe Lys Asn Arg Pro Ala Pro Ser Val 260 265 270Glu Gln Val Lys Thr Cys Leu Lys Glu Tyr Val Cys Asp Ala Tyr Lys 275 280 285Leu Gly Cys His Ser Ala Pro Lys Gln Thr Ile His Val Leu Glu Gln 290 295 300Pro Asp Arg Pro Gln Pro Arg Leu Asp Arg Asn Arg Asp Ser Gly Tyr305 310 315 320Gly Val Ser Val Gly Arg Ile Arg Glu Asp Pro Leu Leu Asp Phe Lys 325 330 335Met Val Val Leu Ser His Asn Thr Ile Ile Gly Ala Ala Gly Ser Gly 340 345 350Val Leu Ile Ala Glu Ile Leu Leu Ala Arg Asn Leu Ile 355 360 36581413DNAPseudomonas aeruginosaDIAMINOBUTYRATE AMINOTRANSFERASE (EctB)DIAMINOBUTYRATE AMINOTRANSFERASE (EctB) 8atggctcacg ttgctacatc agttatcgag gaccaaccct tacgcgccac tcccgcagaa 60ggcgagactc tgtacgagtt ctcccaatct cctctcttag aacgtcagtc tcgccaagag 120agcaatgctc gaagctatcc acgtagaata ccacttgcat taaagaaggc ccgaggtctg 180ctagtggaag acgtcgaagg gaggactttc attgactgtc tcgccggtgc aggaacccta 240gcattgggac ataatcaccc ggtagttata gaagccatta gacaggttct tgctgatgaa 300ttgcccttgc acaccttaga tttgacgacc cctgtgaagg accaattcgt ccaggattta 360tttgggttgc tcccacctgc tttggcagcg gaagccaaga tccaattctg tgggccaaca 420ggaacagacg cagttgaggc cgctcttaag cttgtgcgga cggcgactgg ccgttctaca 480atactaagtt ttcaaggagg atatcacgga atgtcccaag gtgcactggg cttgatgggc 540aacctcggtc caaagaagcc attgggcgca gtactctcaa ccggcgtcca attcctccca 600tacccgtacg attacagatg tccattcggt ctgggtggcg aagctggagt caaggcgaat 660ctacattact tagaaaattt gctcaatgat cctgaaggag gtgtacaatt gccggccgct 720gtcattgttg aagttgtaca aggcgaaggt ggggtcgtgc cagcagattt ggactggtta 780cgaggattac gtaggattac tgagcaggcc ggtgtagccc ttattgtgga tgaaattcaa 840tccggctttg cgcgtactgg tcggatgttc gcctttgagc atgccggcat cgtgcctgat 900gtggtagtgc tttccaaagc tatcggtggc tccctccctt tagctgtggt cgtatatcgg 960gaatggctag acaagtggca acctggggca catgctggaa ccttcagagg caatcagatg 1020gcgatggcag ctggcagtgc agtcatgaga tacctaaaag agcatgatct ggcagcccat 1080gcagccgcca tgggcgaacg actagctgag catctcagga tcttgcagag agactatccg 1140cagttaggtg atatacgagg ccgcggctta atgttaggtg tcgagatagt agatccgcag 1200ggcgaagctg acgctctagg tcatccaccc actgacggag ccctggcctc gcgggtacaa 1260cgggaatgtt tgagaagagg

tctcatattg gaattaggcg ggcgacacgg atctgttgta 1320agattcttgc cacctttgat tattggggca gaacagatag acgaagtggc gcgtagattt 1380gccagggcat taggagctgc ccttgctggg taa 141391266DNAHalomonas elongataDIAMINOBUTYRATE AMINOTRANSFERASE (EctB)DIAMINOBUTYRATE AMINOTRANSFERASE (EctB) 9atgcagacac aaatcttgga acgtatggaa tccgacgtca gaacgtactc aagatctttc 60cctgtagtct ttaccaaggc gcgaaatgct cgacttaccg acgaagaagg gcgagagtac 120atagacttcc tagcaggcgc tggtacgcta aattatgggc ataacaatcc acacctgaag 180caagcattac tcgactacat tgattcagac ggcattgtcc atggtctgga tttctggacc 240gcggcaaagc gcgattacct tgagacactc gaagaggtca ttttgaaacc gcgtggtttg 300gactataagg ttcacttacc gggcccaacg ggcaccaatg cagttgaagc cgccattcgt 360ttggccaggg tcgccaaagg tcgtcataat attgtctctt ttacaaatgg ctttcacggt 420gttactatgg gcgctctggc gacgaccggt aacagaaagt ttcgggaagc gacaggtggc 480gtccctactc aggcagcgag cttcatgcca tttgacgggt atcttggctc ttccactgat 540acacttgatt acttcgaaaa gttattgggt gataagtcag gtgggcttga tgtgccagcc 600gcggtaatag ttgaaacagt ccaaggagaa ggcggaataa acgttgcggg acttgagtgg 660ctcaagagat tagaaagcat ttgtagagca aatgatattt tgttaatcat cgatgatata 720caagccggct gcggaagaac tggaaagttc ttctcattcg aacatgctgg tattactcct 780gatattgtca caaactcgaa atctttgtca ggatatggtt tgccctttgc tcatgtgctt 840atgagaccgg agcttgataa atggaaacca ggacaatata acggaacatt ccggggtttc 900aatctagctt tcgcgaccgc tgctgcagca atgaggaaat actggtcgga cgatacgttt 960gaacgagacg ttcaaaggaa agctagaata gttgaggaaa gatttggtaa aatcgcagct 1020tggctttctg aaaacggtat tgaagcttcc gaacggggta ggggtttgat gagaggtatc 1080gacgttggtt ctggggacat agcagataaa attacgcatc aagcgtttga aaatggttta 1140atcatcgaaa caagtggtca agatggtgag gttgttaaat gcttatgccc cttaaccata 1200ccagatgaag atttagtaga aggtttagac atattagaaa ctagcacaaa acaagccttc 1260tcttaa 126610470PRTPseudomonas aeruginosaDIAMINOBUTYRATE AMINOTRANSFERASE (EctB)DIAMINOBUTYRATE AMINOTRANSFERASE (EctB) 10Met Ala His Val Ala Thr Ser Val Ile Glu Asp Gln Pro Leu Arg Ala1 5 10 15Thr Pro Ala Glu Gly Glu Thr Leu Tyr Glu Phe Ser Gln Ser Pro Leu 20 25 30Leu Glu Arg Gln Ser Arg Gln Glu Ser Asn Ala Arg Ser Tyr Pro Arg 35 40 45Arg Ile Pro Leu Ala Leu Lys Lys Ala Arg Gly Leu Leu Val Glu Asp 50 55 60Val Glu Gly Arg Thr Phe Ile Asp Cys Leu Ala Gly Ala Gly Thr Leu65 70 75 80Ala Leu Gly His Asn His Pro Val Val Ile Glu Ala Ile Arg Gln Val 85 90 95Leu Ala Asp Glu Leu Pro Leu His Thr Leu Asp Leu Thr Thr Pro Val 100 105 110Lys Asp Gln Phe Val Gln Asp Leu Phe Gly Leu Leu Pro Pro Ala Leu 115 120 125Ala Ala Glu Ala Lys Ile Gln Phe Cys Gly Pro Thr Gly Thr Asp Ala 130 135 140Val Glu Ala Ala Leu Lys Leu Val Arg Thr Ala Thr Gly Arg Ser Thr145 150 155 160Ile Leu Ser Phe Gln Gly Gly Tyr His Gly Met Ser Gln Gly Ala Leu 165 170 175Gly Leu Met Gly Asn Leu Gly Pro Lys Lys Pro Leu Gly Ala Val Leu 180 185 190Ser Thr Gly Val Gln Phe Leu Pro Tyr Pro Tyr Asp Tyr Arg Cys Pro 195 200 205Phe Gly Leu Gly Gly Glu Ala Gly Val Lys Ala Asn Leu His Tyr Leu 210 215 220Glu Asn Leu Leu Asn Asp Pro Glu Gly Gly Val Gln Leu Pro Ala Ala225 230 235 240Val Ile Val Glu Val Val Gln Gly Glu Gly Gly Val Val Pro Ala Asp 245 250 255Leu Asp Trp Leu Arg Gly Leu Arg Arg Ile Thr Glu Gln Ala Gly Val 260 265 270Ala Leu Ile Val Asp Glu Ile Gln Ser Gly Phe Ala Arg Thr Gly Arg 275 280 285Met Phe Ala Phe Glu His Ala Gly Ile Val Pro Asp Val Val Val Leu 290 295 300Ser Lys Ala Ile Gly Gly Ser Leu Pro Leu Ala Val Val Val Tyr Arg305 310 315 320Glu Trp Leu Asp Lys Trp Gln Pro Gly Ala His Ala Gly Thr Phe Arg 325 330 335Gly Asn Gln Met Ala Met Ala Ala Gly Ser Ala Val Met Arg Tyr Leu 340 345 350Lys Glu His Asp Leu Ala Ala His Ala Ala Ala Met Gly Glu Arg Leu 355 360 365Ala Glu His Leu Arg Ile Leu Gln Arg Asp Tyr Pro Gln Leu Gly Asp 370 375 380Ile Arg Gly Arg Gly Leu Met Leu Gly Val Glu Ile Val Asp Pro Gln385 390 395 400Gly Glu Ala Asp Ala Leu Gly His Pro Pro Thr Asp Gly Ala Leu Ala 405 410 415Ser Arg Val Gln Arg Glu Cys Leu Arg Arg Gly Leu Ile Leu Glu Leu 420 425 430Gly Gly Arg His Gly Ser Val Val Arg Phe Leu Pro Pro Leu Ile Ile 435 440 445Gly Ala Glu Gln Ile Asp Glu Val Ala Arg Arg Phe Ala Arg Ala Leu 450 455 460Gly Ala Ala Leu Ala Gly465 47011421PRTHalomonas elongataDIAMINOBUTYRATE AMINOTRANSFERASE (EctB)DIAMINOBUTYRATE AMINOTRANSFERASE (EctB) 11Met Gln Thr Gln Ile Leu Glu Arg Met Glu Ser Asp Val Arg Thr Tyr1 5 10 15Ser Arg Ser Phe Pro Val Val Phe Thr Lys Ala Arg Asn Ala Arg Leu 20 25 30Thr Asp Glu Glu Gly Arg Glu Tyr Ile Asp Phe Leu Ala Gly Ala Gly 35 40 45Thr Leu Asn Tyr Gly His Asn Asn Pro His Leu Lys Gln Ala Leu Leu 50 55 60Asp Tyr Ile Asp Ser Asp Gly Ile Val His Gly Leu Asp Phe Trp Thr65 70 75 80Ala Ala Lys Arg Asp Tyr Leu Glu Thr Leu Glu Glu Val Ile Leu Lys 85 90 95Pro Arg Gly Leu Asp Tyr Lys Val His Leu Pro Gly Pro Thr Gly Thr 100 105 110Asn Ala Val Glu Ala Ala Ile Arg Leu Ala Arg Val Ala Lys Gly Arg 115 120 125His Asn Ile Val Ser Phe Thr Asn Gly Phe His Gly Val Thr Met Gly 130 135 140Ala Leu Ala Thr Thr Gly Asn Arg Lys Phe Arg Glu Ala Thr Gly Gly145 150 155 160Val Pro Thr Gln Ala Ala Ser Phe Met Pro Phe Asp Gly Tyr Leu Gly 165 170 175Ser Ser Thr Asp Thr Leu Asp Tyr Phe Glu Lys Leu Leu Gly Asp Lys 180 185 190Ser Gly Gly Leu Asp Val Pro Ala Ala Val Ile Val Glu Thr Val Gln 195 200 205Gly Glu Gly Gly Ile Asn Val Ala Gly Leu Glu Trp Leu Lys Arg Leu 210 215 220Glu Ser Ile Cys Arg Ala Asn Asp Ile Leu Leu Ile Ile Asp Asp Ile225 230 235 240Gln Ala Gly Cys Gly Arg Thr Gly Lys Phe Phe Ser Phe Glu His Ala 245 250 255Gly Ile Thr Pro Asp Ile Val Thr Asn Ser Lys Ser Leu Ser Gly Tyr 260 265 270Gly Leu Pro Phe Ala His Val Leu Met Arg Pro Glu Leu Asp Lys Trp 275 280 285Lys Pro Gly Gln Tyr Asn Gly Thr Phe Arg Gly Phe Asn Leu Ala Phe 290 295 300Ala Thr Ala Ala Ala Ala Met Arg Lys Tyr Trp Ser Asp Asp Thr Phe305 310 315 320Glu Arg Asp Val Gln Arg Lys Ala Arg Ile Val Glu Glu Arg Phe Gly 325 330 335Lys Ile Ala Ala Trp Leu Ser Glu Asn Gly Ile Glu Ala Ser Glu Arg 340 345 350Gly Arg Gly Leu Met Arg Gly Ile Asp Val Gly Ser Gly Asp Ile Ala 355 360 365Asp Lys Ile Thr His Gln Ala Phe Glu Asn Gly Leu Ile Ile Glu Thr 370 375 380Ser Gly Gln Asp Gly Glu Val Val Lys Cys Leu Cys Pro Leu Thr Ile385 390 395 400Pro Asp Glu Asp Leu Val Glu Gly Leu Asp Ile Leu Glu Thr Ser Thr 405 410 415Lys Gln Ala Phe Ser 420121461DNASaccharomyces cerevisiaeHOMOSERINE O-ACETYLTRANSFERASE (MET2; METX)HOMOSERINE O-ACETYLTRANSFERASE (MET2; METX) 12atgtcgcata ctttaaaatc gaaaacgctc caagagctgg acattgagga gattaaggaa 60actaacccat tgctcaaact agttcaaggg cagaggattg ttcaagttcc ggaactagtg 120cttgagtctg gcgtggtcat aaataatttc cctattgctt ataagacgtg gggtacactg 180aatgaagctg gtgataatgt tctggtaatt tgtcatgcct tgactgggtc cgcagatgtt 240gctgactggt ggggccctct tctgggtaac gacttagcat tcgacccatc aaggtttttt 300atcatatgtt taaactctat gggctctcca tatgggtctt tttcgccatt aacgataaat 360gaggagacgg gcgttagata tggacccgaa ttcccattat gtactgtgcg cgatgacgtt 420agagctcaca gaattgttct ggattctctg ggagtaaagt caatagcctg tgttattggt 480ggctctatgg gggggatgct gagtttggaa tgggctgcca tgtatggtaa ggaatatgtg 540aagaatatgg ttgctctggc gacatcagca agacattctg cctggtgcat atcgtggtct 600gaggctcaaa gacaatcgat ttactcagat cccaactact tggacgggta ctatccggta 660gaggagcaac ctgtggccgg actatcggct gcacgtatgt ctgcattgtt gacgtacagg 720acaagaaaca gtttcgagaa caaattctcc agaagatctc cttcaatagc acaacaacaa 780aaagctcaaa gggaggagac acgcaaacca tctactgtca gcgaacactc cctacaaatc 840cacaatgatg ggtataaaac aaaagccagc actgccatcg ctggcatttc tgggcaaaaa 900ggtcaaagcg tggtgtccac cgcatcttct tcggattcat tgaattcttc aacatcgatg 960acttcggtaa gttctgtaac gggtgaagtg aaggacataa agcctgcgca gacgtatttt 1020tctgcacaaa gttacttgag gtaccagggc acaaagttca tcaataggtt cgacgccaat 1080tgttacattg ccatcacacg taaactggat acgcacgatt tggcaagaga cagagtagat 1140gacatcactg aggtcctttc taccatccaa caaccatccc tgatcatcgg tatccaatct 1200gatggactgt tcacatattc agaacaagaa tttttggctg agcacatacc gaagtcgcaa 1260ttagaaaaaa ttgaatctcc cgaaggccac gatgccttcc tattggagtt taagctgata 1320aacaaactga tagtacaatt tttaaaaacc aactgcaagg ccattaccga tgccgctcca 1380agagcttggg gaggtgacgt tggtaacgat gaaacgaaga cgtctgtctt tggtgaggcc 1440gaagaagtta ccaactggta g 146113486PRTSaccharomyces cerevisiaeHOMOSERINE O-ACETYLTRANSFERASE (MET2; METX)HOMOSERINE O-ACETYLTRANSFERASE (MET2; METX) 13Met Ser His Thr Leu Lys Ser Lys Thr Leu Gln Glu Leu Asp Ile Glu1 5 10 15Glu Ile Lys Glu Thr Asn Pro Leu Leu Lys Leu Val Gln Gly Gln Arg 20 25 30Ile Val Gln Val Pro Glu Leu Val Leu Glu Ser Gly Val Val Ile Asn 35 40 45Asn Phe Pro Ile Ala Tyr Lys Thr Trp Gly Thr Leu Asn Glu Ala Gly 50 55 60Asp Asn Val Leu Val Ile Cys His Ala Leu Thr Gly Ser Ala Asp Val65 70 75 80Ala Asp Trp Trp Gly Pro Leu Leu Gly Asn Asp Leu Ala Phe Asp Pro 85 90 95Ser Arg Phe Phe Ile Ile Cys Leu Asn Ser Met Gly Ser Pro Tyr Gly 100 105 110Ser Phe Ser Pro Leu Thr Ile Asn Glu Glu Thr Gly Val Arg Tyr Gly 115 120 125Pro Glu Phe Pro Leu Cys Thr Val Arg Asp Asp Val Arg Ala His Arg 130 135 140Ile Val Leu Asp Ser Leu Gly Val Lys Ser Ile Ala Cys Val Ile Gly145 150 155 160Gly Ser Met Gly Gly Met Leu Ser Leu Glu Trp Ala Ala Met Tyr Gly 165 170 175Lys Glu Tyr Val Lys Asn Met Val Ala Leu Ala Thr Ser Ala Arg His 180 185 190Ser Ala Trp Cys Ile Ser Trp Ser Glu Ala Gln Arg Gln Ser Ile Tyr 195 200 205Ser Asp Pro Asn Tyr Leu Asp Gly Tyr Tyr Pro Val Glu Glu Gln Pro 210 215 220Val Ala Gly Leu Ser Ala Ala Arg Met Ser Ala Leu Leu Thr Tyr Arg225 230 235 240Thr Arg Asn Ser Phe Glu Asn Lys Phe Ser Arg Arg Ser Pro Ser Ile 245 250 255Ala Gln Gln Gln Lys Ala Gln Arg Glu Glu Thr Arg Lys Pro Ser Thr 260 265 270Val Ser Glu His Ser Leu Gln Ile His Asn Asp Gly Tyr Lys Thr Lys 275 280 285Ala Ser Thr Ala Ile Ala Gly Ile Ser Gly Gln Lys Gly Gln Ser Val 290 295 300Val Ser Thr Ala Ser Ser Ser Asp Ser Leu Asn Ser Ser Thr Ser Met305 310 315 320Thr Ser Val Ser Ser Val Thr Gly Glu Val Lys Asp Ile Lys Pro Ala 325 330 335Gln Thr Tyr Phe Ser Ala Gln Ser Tyr Leu Arg Tyr Gln Gly Thr Lys 340 345 350Phe Ile Asn Arg Phe Asp Ala Asn Cys Tyr Ile Ala Ile Thr Arg Lys 355 360 365Leu Asp Thr His Asp Leu Ala Arg Asp Arg Val Asp Asp Ile Thr Glu 370 375 380Val Leu Ser Thr Ile Gln Gln Pro Ser Leu Ile Ile Gly Ile Gln Ser385 390 395 400Asp Gly Leu Phe Thr Tyr Ser Glu Gln Glu Phe Leu Ala Glu His Ile 405 410 415Pro Lys Ser Gln Leu Glu Lys Ile Glu Ser Pro Glu Gly His Asp Ala 420 425 430Phe Leu Leu Glu Phe Lys Leu Ile Asn Lys Leu Ile Val Gln Phe Leu 435 440 445Lys Thr Asn Cys Lys Ala Ile Thr Asp Ala Ala Pro Arg Ala Trp Gly 450 455 460Gly Asp Val Gly Asn Asp Glu Thr Lys Thr Ser Val Phe Gly Glu Ala465 470 475 480Glu Glu Val Thr Asn Trp 48514579DNAHalomonas elongataACID ACETYLTRANSFERASE (EctA)ACID ACETYLTRANSFERASE (EctA) 14atgactccca caacagaaaa ttttactcct agtgcagatc tggctcgccc ttcagtggct 60gacaccgtta ttggctccgc caagaaaaca ctattcatca gaaagcctac cacggacgat 120ggttggggta tctacgagtt agttaaggcg tgcccaccct tggacgtaaa ctctggatac 180gcttacttat tattagccac gcaatttagg gatacgtgtg ctgtcgctac cgacgaggaa 240ggggagatcg ttggctttgt atcaggatac gttaagcgta acgcacctga tacctatttt 300ctatggcaag ttgctgtggg cgaaaaggct cgtgggacgg gtcttgcaag aagattagtc 360gaagccgtat tgatgagacc aggtatggga gatgtccggc acctggagac taccataact 420cctgataacg aagcaagctg gggtctcttt aaacgacttg ccgatagatg gcaagcgcca 480ttgaattcta gggaatattt ctctactggt cagttgggtg gtgaacatga tccggaaaat 540ctggtgagaa ttggaccgtt cgaaccacag caaatttaa 57915192PRTHalomonas elongataACID ACETYLTRANSFERASE (EctA)ACID ACETYLTRANSFERASE (EctA) 15Met Thr Pro Thr Thr Glu Asn Phe Thr Pro Ser Ala Asp Leu Ala Arg1 5 10 15Pro Ser Val Ala Asp Thr Val Ile Gly Ser Ala Lys Lys Thr Leu Phe 20 25 30Ile Arg Lys Pro Thr Thr Asp Asp Gly Trp Gly Ile Tyr Glu Leu Val 35 40 45Lys Ala Cys Pro Pro Leu Asp Val Asn Ser Gly Tyr Ala Tyr Leu Leu 50 55 60Leu Ala Thr Gln Phe Arg Asp Thr Cys Ala Val Ala Thr Asp Glu Glu65 70 75 80Gly Glu Ile Val Gly Phe Val Ser Gly Tyr Val Lys Arg Asn Ala Pro 85 90 95Asp Thr Tyr Phe Leu Trp Gln Val Ala Val Gly Glu Lys Ala Arg Gly 100 105 110Thr Gly Leu Ala Arg Arg Leu Val Glu Ala Val Leu Met Arg Pro Gly 115 120 125Met Gly Asp Val Arg His Leu Glu Thr Thr Ile Thr Pro Asp Asn Glu 130 135 140Ala Ser Trp Gly Leu Phe Lys Arg Leu Ala Asp Arg Trp Gln Ala Pro145 150 155 160Leu Asn Ser Arg Glu Tyr Phe Ser Thr Gly Gln Leu Gly Gly Glu His 165 170 175Asp Pro Glu Asn Leu Val Arg Ile Gly Pro Phe Glu Pro Gln Gln Ile 180 185 19016414DNAHalomonas elongataECTOINE SYNTHASE (EctC)ECTOINE SYNTHASE (EctC) 16atgatagttc gtaacctgga ggaagctagg caaacagata gattagtgac cgctgagaat 60ggcaactggg acagtaccag attatcatta gctgaggacg gaggtaattg ttcttttcac 120attaccagaa tatttgaagg gactgaaact cacatacact acaagcatca ctttgaagcc 180gtttactgca tcgagggtga aggagaagtc gaaaccctcg ctgatggaaa gatctggccc 240ataaaacctg gggatattta tattttggat cagcatgacg aacatttgct tagggcttcg 300aaaactatgc atctagcatg cgtattcacg ccgggtctaa ctggtaatga agttcatcga 360gaagacggtt cctatgcacc agcggatgaa gcagatgatc agaaaccact ttaa 41417137PRTHalomonas elongataECTOINE SYNTHASE (EctC)ECTOINE SYNTHASE (EctC) 17Met Ile Val Arg Asn Leu Glu Glu Ala Arg Gln Thr Asp Arg Leu Val1 5 10 15Thr Ala Glu Asn Gly Asn Trp Asp Ser Thr Arg Leu Ser Leu Ala Glu 20 25 30Asp Gly Gly Asn Cys Ser Phe His Ile Thr Arg Ile Phe Glu Gly Thr 35 40 45Glu Thr His Ile His Tyr Lys His His Phe Glu Ala Val Tyr Cys Ile 50 55 60Glu Gly Glu Gly Glu Val Glu Thr Leu Ala Asp Gly Lys Ile Trp Pro65 70 75 80Ile Lys Pro Gly Asp Ile Tyr Ile Leu Asp Gln His Asp Glu His Leu 85 90 95Leu Arg Ala Ser Lys Thr Met His Leu Ala Cys Val Phe Thr Pro Gly 100 105 110Leu Thr Gly Asn Glu Val His Arg Glu Asp

Gly Ser Tyr Ala Pro Ala 115 120 125Asp Glu Ala Asp Asp Gln Lys Pro Leu 130 135181080DNASaccharomyces cerevisiaeHOMOSERINE DESHYDROGENASE (HOM6)HOMOSERINE DESHYDROGENASE (HOM6) 18atgagcacta aagttgttaa tgttgccgtt atcggtgccg gtgttgttgg ttcagctttc 60ttggatcaat tgttagccat gaagtctacc attacttaca atctagttct tttggctgaa 120gctgagcgtt ctttaatctc caaggacttt tctccattaa atgttggttc tgattggaag 180gctgctttag cagcctccac tactaaaacg ttgcctttgg atgatttaat tgctcatttg 240aagacttcac ctaagccagt cattttggtt gataacactt ccagcgctta cattgctggt 300ttttacacta agtttgtcga aaatggtatt tccattgcta ctccaaacaa gaaggccttt 360tcctctgatt tggctacctg gaaggctctt ttctcaaata agccaactaa cggttttgtc 420tatcatgaag ctaccgtcgg tgctggtttg cctatcatca gtttcttaag agaaattatt 480caaaccggtg acgaagttga aaaaattgaa ggtatcttct ctggtactct atcttatatt 540ttcaacgagt tctccactag tcaagctaac gacgtcaaat tctctgatgt tgtcaaagtt 600gctaaaaaat tgggttatac tgaaccagat ccaagagatg atttgaatgg gttggatgtt 660gctagaaagg ttaccattgt tggtaggata tctggtgtgg aagttgaatc tccaacttcc 720ttccctgtcc agtctttgat tccaaaacca ttggaatctg tcaagtctgc tgatgaattc 780ttggaaaaat tatctgatta cgataaagat ttgactcaat tgaagaagga agctgccact 840gaaaataagg tattgagatt cattggtaaa gtcgatgttg ccaccaaatc tgtgtctgta 900ggaattgaaa agtacgatta ctcacaccca ttcgcatcat tgaagggatc agataacgtt 960atttccatca agactaagcg ttacaccaat cctgttgtca ttcaaggtgc cggtgccggt 1020gctgccgtta ctgccgctgg tgttttgggt gatgttatca agattgctca aagactttaa 108019308PRTSaccharomyces cerevisiaeHOMOSERINE DESHYDROGENASE (HOM6)HOMOSERINE DESHYDROGENASE (HOM6) 19Met Ser Thr Lys Val Val Asn Val Ala Val Ile Gly Ala Gly Val Val1 5 10 15Gly Ser Ala Phe Leu Asp Gln Leu Leu Ala Met Lys Ser Thr Ile Thr 20 25 30Tyr Asn Leu Val Leu Leu Ala Glu Ala Glu Arg Ser Leu Ile Ser Lys 35 40 45Asp Phe Ser Pro Leu Asn Val Gly Ser Asp Trp Lys Ala Ala Leu Ala 50 55 60Ala Ser Thr Thr Lys Thr Leu Pro Leu Asp Asp Leu Ile Ala His Leu65 70 75 80Lys Thr Ser Pro Lys Pro Val Ile Leu Val Asp Asn Thr Ser Ser Ala 85 90 95Tyr Ile Ala Gly Phe Tyr Thr Lys Phe Val Glu Asn Gly Ile Ser Ile 100 105 110Ala Thr Pro Asn Lys Lys Ala Phe Ser Ser Asp Leu Ala Thr Trp Lys 115 120 125Ala Leu Phe Ser Asn Lys Pro Thr Asn Gly Phe Val Tyr His Glu Ala 130 135 140Thr Val Gly Ala Gly Leu Pro Ile Ile Ser Phe Leu Arg Glu Ile Ile145 150 155 160Gln Thr Gly Asp Glu Val Glu Lys Ile Glu Gly Ile Phe Ser Gly Thr 165 170 175Leu Ser Tyr Ile Phe Asn Glu Phe Ser Thr Ser Gln Ala Asn Asp Val 180 185 190Lys Phe Ser Asp Val Val Lys Val Ala Lys Lys Leu Gly Tyr Thr Glu 195 200 205Pro Asp Pro Arg Asp Asp Leu Asn Gly Leu Asp Val Ala Arg Lys Val 210 215 220Thr Ile Val Gly Arg Ile Ser Gly Val Glu Val Glu Ser Pro Thr Ser225 230 235 240Phe Pro Val Gln Ser Leu Ile Pro Lys Pro Leu Glu Ser Val Lys Ser 245 250 255Ala Asp Glu Phe Leu Glu Lys Leu Ser Asp Tyr Asp Lys Asp Leu Thr 260 265 270Gln Leu Lys Lys Glu Ala Ala Thr Glu Asn Lys Val Leu Arg Phe Ile 275 280 285Gly Lys Val Asp Val Ala Thr Lys Ser Val Ser Val Gly Ile Glu Lys 290 295 300Tyr Asp Tyr Ser305201257DNASaccharomyces cerevisiaeASPARTATE TRANSAMINASE (AAT2)ASPARTATE TRANSAMINASE (AAT2) 20atgtctgcca ctctgttcaa taacatcgaa ttgctgcccc ctgatgccct ttttggtatt 60aagcaaaggt acgggcaaga tcaacgtgct accaaggtcg acttgggtat cggggcctac 120agagacgaca acggtaaacc atgggtcttg ccaagtgtta aagccgccga aaagctaatt 180cataacgaca gctcctacaa ccatgaatac ctcggtatta ccggtctgcc aagtttgaca 240tctaacgccg ccaagatcat cttcggtacg caatccgatg cctttcagga agacagagta 300atctcagtac aatcactgtc tggtacgggt gctcttcata tatctgcgaa gtttttttca 360aaattcttcc cagataaact ggtctatttg tctaagccta cttgggccaa ccacatggcc 420atttttgaga atcaaggctt gaaaacggcg acttaccctt actgggccaa cgaaactaag 480tctttggacc taaacggctt tctaaatgct attcaaaaag ctccagaggg ctccattttc 540gttctgcact cttgcgccca taacccaact ggtctggacc ctactagtga acaatgggtt 600caaatcgttg atgctatcgc ctcaaagaac cacatcgcct tatttgacac cgcctaccaa 660gggtttgcca ctggagattt ggacaaggat gcctatgctg tgcgtctagg tgtggagaag 720ctttcaacgg tctctcccgt ctttgtctgt cagtcctttg ccaagaacgc cggtatgtac 780ggtgagcgtg taggttgttt ccatctagca cttacaaaac aagctcaaaa caaaactata 840aagcctgctg ttacatctca attggccaaa atcattcgta gtgaagtgtc caacccaccc 900gcctacggcg ctaagattgt cgctaaactg ttggaaacgc cagaattaac ggaacagtgg 960cacaaggata tggttaccat gtcctccaga attacgaaaa tgaggcacgc attaagagac 1020catttagtca agttgggcac tcctggcaac tgggatcata tagtaaatca atgcgggatg 1080ttctccttta cagggttgac tcctcaaatg gttaaacgac ttgaagaaac ccacgcagtt 1140tacttggttg cctcaggtag agcttctatt gctggattga atcaaggaaa cgtggaatac 1200gtggctaaag ccattgatga agtggtgcgc ttctatacta ttgaagctaa attgtaa 125721418PRTSaccharomyces cerevisiaeASPARTATE TRANSAMINASE (AAT2)ASPARTATE TRANSAMINASE (AAT2) 21Met Ser Ala Thr Leu Phe Asn Asn Ile Glu Leu Leu Pro Pro Asp Ala1 5 10 15Leu Phe Gly Ile Lys Gln Arg Tyr Gly Gln Asp Gln Arg Ala Thr Lys 20 25 30Val Asp Leu Gly Ile Gly Ala Tyr Arg Asp Asp Asn Gly Lys Pro Trp 35 40 45Val Leu Pro Ser Val Lys Ala Ala Glu Lys Leu Ile His Asn Asp Ser 50 55 60Ser Tyr Asn His Glu Tyr Leu Gly Ile Thr Gly Leu Pro Ser Leu Thr65 70 75 80Ser Asn Ala Ala Lys Ile Ile Phe Gly Thr Gln Ser Asp Ala Phe Gln 85 90 95Glu Asp Arg Val Ile Ser Val Gln Ser Leu Ser Gly Thr Gly Ala Leu 100 105 110His Ile Ser Ala Lys Phe Phe Ser Lys Phe Phe Pro Asp Lys Leu Val 115 120 125Tyr Leu Ser Lys Pro Thr Trp Ala Asn His Met Ala Ile Phe Glu Asn 130 135 140Gln Gly Leu Lys Thr Ala Thr Tyr Pro Tyr Trp Ala Asn Glu Thr Lys145 150 155 160Ser Leu Asp Leu Asn Gly Phe Leu Asn Ala Ile Gln Lys Ala Pro Glu 165 170 175Gly Ser Ile Phe Val Leu His Ser Cys Ala His Asn Pro Thr Gly Leu 180 185 190Asp Pro Thr Ser Glu Gln Trp Val Gln Ile Val Asp Ala Ile Ala Ser 195 200 205Lys Asn His Ile Ala Leu Phe Asp Thr Ala Tyr Gln Gly Phe Ala Thr 210 215 220Gly Asp Leu Asp Lys Asp Ala Tyr Ala Val Arg Leu Gly Val Glu Lys225 230 235 240Leu Ser Thr Val Ser Pro Val Phe Val Cys Gln Ser Phe Ala Lys Asn 245 250 255Ala Gly Met Tyr Gly Glu Arg Val Gly Cys Phe His Leu Ala Leu Thr 260 265 270Lys Gln Ala Gln Asn Lys Thr Ile Lys Pro Ala Val Thr Ser Gln Leu 275 280 285Ala Lys Ile Ile Arg Ser Glu Val Ser Asn Pro Pro Ala Tyr Gly Ala 290 295 300Lys Ile Val Ala Lys Leu Leu Glu Thr Pro Glu Leu Thr Glu Gln Trp305 310 315 320His Lys Asp Met Val Thr Met Ser Ser Arg Ile Thr Lys Met Arg His 325 330 335Ala Leu Arg Asp His Leu Val Lys Leu Gly Thr Pro Gly Asn Trp Asp 340 345 350His Ile Val Asn Gln Cys Gly Met Phe Ser Phe Thr Gly Leu Thr Pro 355 360 365Gln Met Val Lys Arg Leu Glu Glu Thr His Ala Val Tyr Leu Val Ala 370 375 380Ser Gly Arg Ala Ser Ile Ala Gly Leu Asn Gln Gly Asn Val Glu Tyr385 390 395 400Val Ala Lys Ala Ile Asp Glu Val Val Arg Phe Tyr Thr Ile Glu Ala 405 410 415Lys Leu221317DNAEntodinium caudatumGLUTAMATE DESHYDROGENASE (GDH)GLUTAMATE DESHYDROGENASE (GDH) 22atgatagatt tagaagcgag aaaccctgct caacccgaat tcattcaagc cagtagagaa 60gtaatcgaat cgatcattga tgttgttaat agcaatccga aatacctgga aaacaaaatt 120ttggagagaa ttacggaacc aaacctaatt cacgaattca aagtcgaatg ggagaatgac 180aagcacgaaa tcatggtgaa caaaggttat cgtattcagt tcaataatgc gataggtccc 240tataagggag gcctaaggtt tcacagagca gtcactctag gtactctgaa attccttggt 300tttgaacaga tatttaagaa ttccttgaca ggattaccta tgggaggtgg caaaggtggt 360tcagattttg atcctagagg taaatcagat gccgagattt taagattctg taggtctttt 420atgacttcgt tgttcaaata tattgggcca gagatagatg ttcctgctgg agatataggt 480gtcggaggta gggaaattgg ttacttgttt ggccaataca aaagactgac ccaacaacat 540gaaggagttc taactggtaa gggtcttaac tggggtggct ctcttgttag acctgaagcc 600acaggttttg gaacgatgta ttttgctaac gaagtcttac atgcacatgg tgacgacatc 660aaggggaaaa ccattgccat atccggattt ggtaatgttg cctttggtgc tgtcttaaaa 720gcgaaacaat taggcgctaa ggtagtcact atatctggcc cagatggtta catttatgac 780gagaatggga taaacaccga cgagaaaatc aactacatgt tggaattaag agcctcaaat 840aatgatgtgg ttgcgccatt tgcagagaag tttggtgcaa aattcatacc agggaagaag 900ccatgggaag ttccagtgga tatggctttt ccctgtgcca ttcagaacga attgaatgcc 960gaagatgctg ccactttaca taagaatgga gtgaaatatg tgatcgagac atccaatatg 1020ggctgtacag cagatgctgt gcaatacttc attaagaacc gtattgtttt cgctccgggt 1080aaagcagcta atgctggtgg tgttgcagta tctgggttgg aaatgagcca aaactcaatg 1140aagttgaact ggacagctga agaagttgac gctaaattga agaatatcat gaccaatatt 1200catgcaagtt gcgtaaagga aggaaaagag agtgacgggt atatcaatta cgttaaaggc 1260gcaaatatag caggcttcaa gaaagtagct gatgcaatgg tagatcttgg ctattaa 131723438PRTEntodinium caudatumGLUTAMATE DESHYDROGENASE (GDH)GLUTAMATE DESHYDROGENASE (GDH) 23Met Ile Asp Leu Glu Ala Arg Asn Pro Ala Gln Pro Glu Phe Ile Gln1 5 10 15Ala Ser Arg Glu Val Ile Glu Ser Ile Ile Asp Val Val Asn Ser Asn 20 25 30Pro Lys Tyr Leu Glu Asn Lys Ile Leu Glu Arg Ile Thr Glu Pro Asn 35 40 45Leu Ile His Glu Phe Lys Val Glu Trp Glu Asn Asp Lys His Glu Ile 50 55 60Met Val Asn Lys Gly Tyr Arg Ile Gln Phe Asn Asn Ala Ile Gly Pro65 70 75 80Tyr Lys Gly Gly Leu Arg Phe His Arg Ala Val Thr Leu Gly Thr Leu 85 90 95Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ser Leu Thr Gly Leu 100 105 110Pro Met Gly Gly Gly Lys Gly Gly Ser Asp Phe Asp Pro Arg Gly Lys 115 120 125Ser Asp Ala Glu Ile Leu Arg Phe Cys Arg Ser Phe Met Thr Ser Leu 130 135 140Phe Lys Tyr Ile Gly Pro Glu Ile Asp Val Pro Ala Gly Asp Ile Gly145 150 155 160Val Gly Gly Arg Glu Ile Gly Tyr Leu Phe Gly Gln Tyr Lys Arg Leu 165 170 175Thr Gln Gln His Glu Gly Val Leu Thr Gly Lys Gly Leu Asn Trp Gly 180 185 190Gly Ser Leu Val Arg Pro Glu Ala Thr Gly Phe Gly Thr Met Tyr Phe 195 200 205Ala Asn Glu Val Leu His Ala His Gly Asp Asp Ile Lys Gly Lys Thr 210 215 220Ile Ala Ile Ser Gly Phe Gly Asn Val Ala Phe Gly Ala Val Leu Lys225 230 235 240Ala Lys Gln Leu Gly Ala Lys Val Val Thr Ile Ser Gly Pro Asp Gly 245 250 255Tyr Ile Tyr Asp Glu Asn Gly Ile Asn Thr Asp Glu Lys Ile Asn Tyr 260 265 270Met Leu Glu Leu Arg Ala Ser Asn Asn Asp Val Val Ala Pro Phe Ala 275 280 285Glu Lys Phe Gly Ala Lys Phe Ile Pro Gly Lys Lys Pro Trp Glu Val 290 295 300Pro Val Asp Met Ala Phe Pro Cys Ala Ile Gln Asn Glu Leu Asn Ala305 310 315 320Glu Asp Ala Ala Thr Leu His Lys Asn Gly Val Lys Tyr Val Ile Glu 325 330 335Thr Ser Asn Met Gly Cys Thr Ala Asp Ala Val Gln Tyr Phe Ile Lys 340 345 350Asn Arg Ile Val Phe Ala Pro Gly Lys Ala Ala Asn Ala Gly Gly Val 355 360 365Ala Val Ser Gly Leu Glu Met Ser Gln Asn Ser Met Lys Leu Asn Trp 370 375 380Thr Ala Glu Glu Val Asp Ala Lys Leu Lys Asn Ile Met Thr Asn Ile385 390 395 400His Ala Ser Cys Val Lys Glu Gly Lys Glu Ser Asp Gly Tyr Ile Asn 405 410 415Tyr Val Lys Gly Ala Asn Ile Ala Gly Phe Lys Lys Val Ala Asp Ala 420 425 430Met Val Asp Leu Gly Tyr 43524554DNASaccharomyces cerevisiaepTDH3pTDH3 24ccaaaatagg gggcgggtta cacagaatat ataacatcgt aggtgtctgg gtgaacagtt 60tattcctggc atccactaaa tataatggag cccgcttttt aagctggcat ccagaaaaaa 120aaagaatccc agcaccaaaa tattgttttc ttcaccaacc atcagttcat aggtccattc 180tcttagcgca actacagaga acaggggcac aaacaggcaa aaaacgggca caacctcaat 240ggagtgatgc aacctgcctg gagtaaatga tgacacaagg caattgaccc acgcatgtat 300ctatctcatt ttcttacacc ttctattacc ttctgctctc tctgatttgg aaaaagctga 360aaaaaaaggt tgaaaccagt tccctgaaat tattccccta cttgactaat aagtatataa 420agacggtagg tattgattgt aattctgtaa atctatttct taaacttctt aaattctact 480tttatagtta gtcttttttt tagttttaaa acaccaagaa cttagtttcg aataaacaca 540cataaacaaa caaa 55425550DNASaccharomyces cerevisiaepENO2pENO2 25cgctcagcat ctgcttcttc ccaaagatga acgcggcgtt atgtcactaa cgacgtgcac 60caacttgcgg aaagtggaat cccgttccaa aactggcatc cactaattga tacatctaca 120caccgcacgc cttttttctg aagcccactt tcgtggactt tgccatatgc aaaattcatg 180aagtgtgata ccaagtcagc atacacctca ctagggtagt ttctttggtt gtattgatca 240tttggttcat cgtggttcat taattttttt tctccattgc tttctggctt tgatcttact 300atcatttgga tttttgtcga aggttgtaga attgtatgtg acaagtggca ccaagcatat 360ataaaaaaaa aaagcattat cttcctacca gagttgattg ttaaaaacgt atttatagca 420aacgcaattg taattaattc ttattttgta tcttttcttc ccttgtctca atcttttatt 480tttattttat ttttcttttc ttagtttctt tcataacacc aagcaactaa tactataaca 540tacaataata 55026419DNASaccharomyces cerevisiaepTEF KlpTEF Kl 26ctctctcgca ataacaatga acactgggtc aatcatagcc tacacaggtg aacagagtag 60cgtttataca gggtttatac ggtgattcct acggcaaaaa tttttcattt ctaaaaaaaa 120aaagaaaaat ttttctttcc aacgctagaa ggaaaagaaa aatctaatta aattgatttg 180gtgattttct gagagttccc tttttcatat atcgaatttt gaatataaaa ggagatcgaa 240aaaatttttc tattcaatct gttttctggt tttatttgat agtttttttg tgtattatta 300ttatggatta gtactggttt atatgggttt ttctgtataa cttcttttta ttttagtttg 360tttaatctta ttttgagtta cattatagtt ccctaactgc aagagaagta acattaaaa 41927598DNASaccharomyces cerevisiaepTEF3pTEF3 27ggctgataat agcgtataaa caatgcatac tttgtacgtt caaaatacaa tgcagtagat 60atatttatgc atattacata taatacatat cacataggaa gcaacaggcg cgttggactt 120ttaattttcg aggaccgcga atccttacat cacacccaat cccccacaag tgatccccca 180cacaccatag cttcaaaatg tttctactcc ttttttactc ttccagattt tctcggactc 240cgcgcatcgc cgtaccactt caaaacaccc aagcacagca tactaaattt cccctctttc 300ttcctctagg gtgtcgttaa ttacccgtac taaaggtttg gaaaagaaaa aagagaccgc 360ctcgtttctt tttcttcgtc gaaaaaggca ataaaaattt ttatcacgtt tctttttctt 420gaaaattttt ttttttgatt tttttctctt tcgatgacct cccattgata tttaagttaa 480taaacggtct tcaatttctc aagtttcagt ttcatttttc ttgttctatt acaacttttt 540ttacttcttg ctcattagaa agaaagcata gcaatctaat ctaagtttta attacaaa 59828383DNASaccharomyces cerevisiaepTEF1pTEF1 28gtttagcttg cctcgtcccc gccgggtcac ccggccagcg acatggaggc ccagaatacc 60ctccttgaca gtcttgacgt gcgcagctca ggggcatgat gtgactgtcg cccgtacatt 120tagcccatac atccccatgt ataatcattt gcatccatac attttgatgg ccgcacggcg 180cgaagcaaaa attacggctc ctcgctgcag acctgcgagc agggaaacgc tcccctcaca 240gacgcgttga attgtcccca cgccgcgccc ctgtagagaa atataaaagg ttaggatttg 300ccactgaggt tcttctttca tatacttcct tttaaaatct tgctacgata cagttctcac 360atcacatccg aacataaaca acc 38329700DNASaccharomyces cerevisiaepADH1pADH1 29gggtgtacaa tatggacttc ctcttttctg gcaaccaaac ccatacatcg ggattcctat 60aataccttcg ttggtctccc taacatgtag gtggcggagg ggagatatac aatagaacag 120ataccagaca agacataatg ggctaaacaa gactacacca attacactgc ctcattgatg 180gtggtacata acgaactaat actgtagccc tagacttgat agccatcatc atatcgaagt 240ttcactaccc tttttccatt tgccatctat tgaagtaata ataggcgcat gcaacttctt 300ttcttttttt ttcttttctc tctcccccgt tgttgtctca ccatatccgc aatgacaaaa 360aaatgatgga agacactaaa ggaaaaaatt aacgacaaag acagcaccaa cagatgtcgt 420tgttccagag ctgatgaggg gtatctcgaa gcacacgaaa ctttttcctt ccttcattca 480cgcacactac tctctaatga gcaacggtat acggccttcc ttccagttac ttgaatttga 540aataaaaaaa agtttgctgt cttgctatca agtataaata gacctgcaat tattaatctt 600ttgtttcctc gtcattgttc tcgttccctt tcttccttgt ttctttttct gcacaatatt 660tcaagctata ccaagcatac aatcaactat ctcatataca 70030549DNASaccharomyces cerevisiaepGPM 1pGPM 1 30gccaaacttt tcggttaaca catgcagtga tgcacgcgcg atggtgctaa gttacatata

60tatatatata tatatatata tatatatata gccatagtga tgtctaagta acctttatgg 120tatatttctt aatgtggaaa gatactagcg cgcgcaccca cacacaagct tcgtcttttc 180ttgaagaaaa gaggaagctc gctaaatggg attccacttt ccgttccctg ccagctgatg 240gaaaaaggtt agtggaacga tgaagaataa aaagagagat ccactgaggt gaaatttcag 300ctgacagcga gtttcatgat cgtgatgaac aatggtaacg agttgtggct gttgccaggg 360agggtggttc tcaactttta atgtatggcc aaatcgctac ttgggtttgt tatataacaa 420agaagaaata atgaactgat tctcttcctc cttcttgtcc tttcttaatt ctgttgtaat 480taccttcctt tgtaattttt tttgtaatta ttcttcttaa taatccaaac aaacacacat 540attacaata 54931650DNASaccharomyces cerevisiaepFBA1pFBA1 31acgcaagccc taagaaatga ataacaatac tgacagtact aaataattgc ctacttggct 60tcacatacgt tgcatacgtc gatatagata ataatgataa tgacagcagg attatcgtaa 120tacgtaatag ttgaaaatct caaaaatgtg tgggtcatta cgtaaataat gataggaatg 180ggattcttct atttttcctt tttccattct agcagccgtc gggaaaacgt ggcatcctct 240ctttcgggct caattggagt cacgctgccg tgagcatcct ctctttccat atctaacaac 300tgagcacgta accaatggaa aagcatgagc ttagcgttgc tccaaaaaag tattggatgg 360ttaataccat ttgtctgttc tcttctgact ttgactcctc aaaaaaaaaa aatctacaat 420caacagatcg cttcaattac gccctcacaa aaactttttt ccttcttctt cgcccacgtt 480aaattttatc cctcatgttg tctaacggat ttctgcactt gatttattat aaaaagacaa 540agacataata cttctctatc aatttcagtt attgttcttc cttgcgttat tcttctgttc 600ttctttttct tttgtcatat ataaccataa ccaagtaata catattcaaa 65032700DNASaccharomyces cerevisiaepPDC1pPDC1 32ttatttacct atctctaaac ttcaacacct tatatcataa ctaatatttc ttgagataag 60cacactgcac ccataccttc cttaaaaacg tagcttccag tttttggtgg ttccggcttc 120cttcccgatt ccgcccgcta aacgcatatt tttgttgcct ggtggcattt gcaaaatgca 180taacctatgc atttaaaaga ttatgtatgc tcttctgact tttcgtgtga tgaggctcgt 240ggaaaaaatg aataatttat gaatttgaga acaattttgt gttgttacgg tattttacta 300tggaataatc aatcaattga ggattttatg caaatatcgt ttgaatattt ttccgaccct 360ttgagtactt ttcttcataa ttgcataata ttgtccgctg cccctttttc tgttagacgg 420tgtcttgatc tacttgctat cgttcaacac caccttattt tctaactatt ttttttttag 480ctcatttgaa tcagcttatg gtgatggcac atttttgcat aaacctagct gtcctcgttg 540aacataggaa aaaaaaatat ataaacaagg ctctttcact ctccttgcaa tcagatttgg 600gtttgttccc tttattttca tatttcttgt catattcctt tctcaattat tattttctac 660tcataacctc acgcaaaata acacagtcaa atcaatcaaa 70033998DNASaccharomyces cerevisiaepCCW12pCCW12 33aaccagggca aagcaaaata aaagaaactt aatacgttat gccgtaatga agggctacca 60aaaacgataa tctcaactgt aaacaggtac aatgcggacc cttttgccac aaaacataca 120tcattcattg ccggaaaaag aaagaagtga agacagcagt gcagccagcc atgttgcgcc 180aatctaatta tagatgctgg tgccctgagg atgtatctgg agccagccat ggcatcatgc 240gctaccgccg gatgtaaaat ccgacacgca aaagaaaacc ttcgaggttg cgcacttcgc 300ccacccatga accacacggt tagtccaaaa ggggcagttc agattccaga tgcgggaatt 360agcttgctgc caccctcacc tcactaacgc tgcggtgtgc ggatacttca tgctatttat 420agacgcgcgt gtcggaatca gcacgcgcaa gaaccaaatg ggaaaatcgg aatgggtcca 480gaactgcttt gagtgctggc tattggcgtc tgatttccgt tttgggaatc ctttgccgcg 540cgcccctctc aaaactccgc acaagtccca gaaagcggga aagaaataaa acgccaccaa 600aaaaaaaaat aaaagccaat cctcgaagcg tgggtggtag gccctggatt atcccgtaca 660agtatttctc aggagtaaaa aaaccgtttg ttttggaatt ccccatttcg cggccaccta 720cgccgctatc tttgcaacaa ctatctgcga taactcagca aattttgcat attcgtgttg 780cagtattgcg ataatgggag tcttacttcc aacataacgg cagaaagaaa tgtgagaaaa 840ttttgcatcc tttgcctccg ttcaagtata taaagtcggc atgcttgata atctttcttt 900ccatcctaca ttgttctaat tattcttatt ctcctttatt ctttcctaac ataccaagaa 960attaatcttc tgtcattcgc ttaaacacta tatcaata 99834700DNASaccharomyces cerevisiaepGK1pGK1 34gtgagtaagg aaagagtgag gaactatcgc atacctgcat ttaaagatgc cgatttgggc 60gcgaatcctt tattttggct tcaccctcat actattatca gggccagaaa aaggaagtgt 120ttccctcctt cttgaattga tgttaccctc ataaagcacg tggcctctta tcgagaaaga 180aattaccgtc gctcgtgatt tgtttgcaaa aagaacaaaa ctgaaaaaac ccagacacgc 240tcgacttcct gtcttcctat tgattgcagc ttccaatttc gtcacacaac aaggtcctag 300cgacggctca caggttttgt aacaagcaat cgaaggttct ggaatggcgg gaaagggttt 360agtaccacat gctatgatgc ccactgtgat ctccagagca aagttcgttc gatcgtactg 420ttactctctc tctttcaaac agaattgtcc gaatcgtgtg acaacaacag cctgttctca 480cacactcttt tcttctaacc aagggggtgg tttagtttag tagaacctcg tgaaacttac 540atttacatat atataaactt gcataaattg gtcaatgcaa gaaatacata tttggtcttt 600tctaattcgt agtttttcaa gttcttagat gctttctttt tctctttttt acagatcatc 660aaggaagtaa ttatctactt tttacaacaa atataaaaca 70035913DNASaccharomyces cerevisiaepMDH2pMDH2 35ccttcgctaa ataataaacc tgaactgtac ttagcgaagc cttcatagca cctacgtaca 60cgtatatata gacattttac gtaatggaga aactgaggtt tttgttttca ctttttttct 120ttctttttca ctattgctcg aaccgcctgc gatgagctaa gaaaaaaaag tgaaagaaat 180catagaaagc aaaaatgaga ttatatagcc cagagccctc ttctggcgcc tgtcccaagg 240cggaccaaca acaacacttg cccaaaccta agaaaatccc ctcatacttt tccgtttgta 300tctcctactt tcttacttcc tttttttctt ctttatttgc ttggtttacc attgaagtcc 360atttttacta cagacaatag ctagtcattc gctatcttcc gtttgtcact ttttttcaaa 420tttctcatct atatagcgaa gtacggaaaa gatgtcactt gccggcatct cggccttccc 480cggccaaatg gactcatcat ctacgatacg gcccctttaa tccgcaatta ctttgcccat 540tcggccgtag ccgttctaaa gccgccgtgc cttgccccca atactcccct aatgatccgg 600gaagttccgg tttttttcct ttgtttagtg gcattttgtg ttgcccaagg ttgggaaggt 660ccgatttgac tttaaggaac tacggaaggt atctaaggtt tctaaaaaca atatacacgc 720gcgtgcgtag atatataaag ataaagattt atcgatatga gataaagatt gctgcatgat 780tctccttctg attctttttc cctgtatata ttttctcccc ttctgtataa atcgtacagt 840cagaagtagt ccagaatata gtgctgcaga ctattacaaa agttcaatac aatatcataa 900aagttatagt aac 91336406DNASaccharomyces cerevisiaepURA3pURA3 36ggtacccaaa ccgaagttat ctgatgtaga aaaggattaa agatgctaag agatagtgat 60gatatttcat aaataatgta attctatata tgttaattac cttttttgcg aggcatattt 120atggtgaagg ataagttttg accatcaaag aaggttaatg tggctgtggt ttcagggtcc 180ataaagcttt tcaattcatc tttttttttt ttgttctttt ttttgattcc ggtttctttg 240aaattttttt gattcggtaa tctccgagca gaaggaagaa cgaaggaagg agcacagact 300tagattggta tatatacgca tatgtggtgt tgaagaaaca tgaaattgcc cagtattctt 360aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatc 40637535DNASaccharomyces cerevisiaepRPLA1pRPLA1 37tcaagttgga tactgatctg atctctccgc cctactacca gggaccctca tgattaccgc 60tcgaatgcga cgtttcctgc ctcataaaac tggcttgaaa atatttattc gctgaacagt 120agcctagctt ataaaaattt catttaatta atgtaatatg aaaactcaca tgccttctgt 180ttctaaaatt gtcacagcaa gaaataacat taccatacgt gatcttatta aactctagta 240tcttgtctaa tacttcattt aaaagaagcc ttaaccctgt agcctcatct atgtctgcta 300catatcgtga ggtacgaata tcgtaagatg ataccacgca actttgtaat gatttttttt 360ttttcatttt ttaaagaatg cctttacatg gtatttgaaa aaaatatctt tataaagttt 420gcgatctctt ctgttctgaa taatttttag taaaagaaat caaaagaata aagaaatagt 480ccgctttgtc caatacaaca gcttaaaccg attatctcta aaataacaag aagaa 53538400DNASaccharomyces cerevisiaepSAM4pSAM4 38agattttggt gttagatggt actcttgcat atgtaacctt taataaattt tgcaaatcga 60attcctttgt aacgtgcaaa gcattttata gcctggcgct cgcattgtta agcaacaggc 120ggtgcggcaa cgttgaaatg tttcacgcag ggttttttac gtactgcacg gcattctgga 180gtgaaaaaaa atgaaaagta cagctcgaag ttttttgtcc atcggttgta ctttgcagag 240tattagtcat ttttgatatc agagtactac tatcgaagca tttttacgct tgaataactt 300gaatattatt gaaagcttag ttcaaccaag ctgaaaagaa ccattattca acataattgg 360aaatcatttc gttactaaat cgtccgaaaa ttgcagaaaa 40039505DNASaccharomyces cerevisiaepCUP1-1pCUP1-1 39cggcaaactt caacgatttc tatgatgcat tttataatta gtaagccgat cccattaccg 60acatttgggc gctatacgtg catatgttca tgtatgtatc tgtatttaaa acacttttgt 120attatttttc ctcatatatg tgtataggtt tatacggatg atttaattat tacttcacca 180ccctttattt caggctgata tcttagcctt gttactagtt agaaaaagac atttttgctg 240tcagtcactg tcaagagatt cttttgctgg catttcttcc agaagcaaaa agagcgatgc 300gtcttttccg ctgaaccgtt ccagcaaaaa agactaccaa cgcaatatgg attgtcagaa 360tcatataaaa gagaagcaaa taactccttg tcttgtatca attgcattat aatatcttct 420tgttagtgca atatcatata gaagtcatcg aaatagatat taagaaaaac aaactgtaca 480atcaatcaat caatcatcac ataaa 50540500DNACandida glabratapCUP1.CglapCUP1.Cgla 40cacaccacac aaccgtcagc accccggctg tacgtctgtg aaggctgcgg tatagacacg 60gactgcgata cagaactcat gacttatatc tgtagactcc tctgcttcaa tgcgaactcc 120aggatcaccg aatagcatgc gatgagctgt tgattcttat atataattat ctattgcatt 180ttttttttaa tgctgcatgg gggggcctag taaatcaccc gtacaagtca cgcgtgagag 240aaagagaagg gccctttcgt cgtggaagcg tggatcgtga gcgacctgtt tctaaatata 300gcttttgggt aggatattat attaagtgaa attttattag agggtaaatg tatgtgaaag 360ttatgtataa tatgttgcta aattagcgat cgtgaatgca tagaatctaa tcgttataga 420aaaccgcaac ttgtgctgtt ttgttgtgtt ttcttgtcgt ttttttatat tatttatcta 480gtattttgct ttagttgtta 50041425DNASaccharomyces bayanuspCUP1.SbapCUP1.Sba 41agaaggaggg gtcctattac caatacttgg acgctatacg tgcatatgta catgtacgta 60tctgtattta aacacttttg tattattttc tttatatatg tgtataggtt tacatggttg 120acttttatca ttgtttgtgc acatttgcaa tggccatttt tttgtttttg agaaaggtat 180tattgctgtc actattcgag atgcttttgc tgacattcct cctagaagcc aaaaggccga 240tgcgtttttt ccgctgagag gataccagca aaaaaagcta ccagtacaag atgggacggc 300aaaagcgtat aaaagaagaa gcaaaatgac cagatatgct ttcaatttca tcaatgtttc 360tttctccctg ttatgatcca gaagaataat caaaagcaaa acatctattc aatcaatctc 420ataaa 42542741DNAArtificial SequenceSynthetic pACU1Synthetic pACU1 42ttacattatc aatccttgcg tttcagcttc cactaattta gatgactatt tctcatcatt 60tgcgtcatct tctaacaccg tatatgataa tatactagta acgtaaatac tagttagtag 120atgatagttg atttttattc caacactaag aaataatttc gccatttctt gaatgtattt 180aaagatattt aatgctataa tagacattta aatccaattc ttccaacata caatgggagt 240ttggccgagt ggtttaaggc gtcagattta ggtggattta acctctaaaa tctctgatat 300cttcggatgc aagggttcga atcccttagc tctcattatt ttttgctttt tctcttgaat 360tcgaaaaaga catttttgct gtcagtcact gtcaagagat tcttttgctg gcatttcttc 420cagaagcaaa aagagcgatg cgtcttttcc gctgaaccgt tccagcaaaa aagactacca 480acgaattcta attaagttag tcaaggcgcc atcctcatga aaactgtgta acataataac 540cgaagtgtcg aaaaggtggc accttgtcca attgaacacg ctcgatgaaa aaaataagat 600atatataagg ttaagtaaag cgtctgttag aaaggaagtt tttccttttt cttgctctct 660tgtcttttca tctactattt ccttcgtgta atacagggtc gtcagataca tagatacaat 720tctattaccc ccatccatac a 74143757DNAArtificial SequenceSynthetic pACU2Synthetic pACU2 43ttacattatc aatccttgcg tttcagcttc cactaattta gatgactatt tctcatcatt 60tgcgtcatct tctaacaccg tatatgataa tatactagta acgtaaatac tagttagtag 120atgatagttg atttttattc caacactaag aaataatttc gccatttctt gaatgtattt 180aaagatattt aatgctataa tagacattta aatccaattc ttccaacata caatgggagt 240ttggccgagt ggtttaaggc gtcagattta ggtggattta acctctaaaa tctctgatat 300cttcggatgc aagggttcga atcccgaatt cgaaaaagac atttttgctg tcagtcactg 360tcaagagatt cttttgctgg catttcttcc agaagcaaaa agagcgatgc gtcttttccg 420ctgaaccgtt ccagcaaaaa agactaccaa cgaattccac gtgaagctgt cgatattggg 480gaactgtggt ggttggcaaa tgactaatta agttagtcaa ggcgccatcc tcatgaaaac 540tgtgtaacat aataaccgaa gtgtcgaaaa ggtggcacct tgtccaattg aacacgctcg 600atgaaaaaaa taagatatat ataaggttaa gtaaagcgtc tgttagaaag gaagtttttc 660ctttttcttg ctctcttgtc ttttcatcta ctatttcctt cgtgtaatac agggtcgtca 720gatacataga tacaattcta ttacccccat ccataca 75744498DNAArtificial SequenceSynthetic pACU3pSynthetic pACU3p 44ttatattgaa ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag aagtttaata 60atcatattac atggcattac caccatatac atatccatat ctaatcttac ttatatgttg 120tggaaatgta aagagcccga attcgaaaaa gacatttttg ctgtcagtca ctgtcaagag 180attcttttgc tggcatttct tccagaagca aaaagagcga tgcgtctttt ccgctgaacc 240gttccagcaa aaaagactac caacgaattc ggatgataat gcgattagtt ttttagcctt 300atttctgggg taattaatca gcgaagcgat gatttttgat ctattaacag atatataaat 360ggaaaagctg cataaccact ttaactaata ctttcaacat tttcagtttg tattacttct 420tattcaaatg tcataaaagt atcaacaaaa aattgttaat atacctctat actttaacgt 480caaggagaaa aaactata 49845498DNAArtificial SequenceSynthetic pACU4pSynthetic pACU4p 45ttatattgaa ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag aagtttaata 60atcatattac atggcattac caccatatac atatccatat ctaatcttac ttatatgttg 120tggaaatgta aagagcccga attcgttggt agtctttttt gctggaacgg ttcagcggaa 180aagacgcatc gctctttttg cttctggaag aaatgccagc aaaagaatct cttgacagtg 240actgacagca aaaatgtctt tttcgaattc ggatgataat gcgattagtt ttttagcctt 300atttctgggg taattaatca gcgaagcgat gatttttgat ctattaacag atatataaat 360ggaaaagctg cataaccact ttaactaata ctttcaacat tttcagtttg tattacttct 420tattcaaatg tcataaaagt atcaacaaaa aattgttaat atacctctat actttaacgt 480caaggagaaa aaactata 49846530DNAArtificial SequenceSynthetic pACU5Synthetic pACU5 46ggaggacgaa acaaaaaagt gaaaaaaaat gaaaattttt ttggaaaacc aagaaatgaa 60ttatatttcc gtgtgagacg acatcgtcga atatgattca gggtaacagt attgatgtaa 120tcaatttcct acctgaatct aaaattcccg gaattcgaaa aagacatttt tgctgtcagt 180cactgtcaag agattctttt gctggcattt cttccagaag caaaaagagc gatgcgtctt 240ttccgctgaa ccgttccagc aaaaaagact accaacgaat tccgagcaga tccgccaggc 300gtgtatatat agcgtggatg gccaggcaac tttagtgctg acacatacag gcatatatat 360atgtgtgcga cgacacatga tcatatggca tgcatgtgct ctgtatgtat ataaaactct 420tgttttcttc ttttctctaa atattctttc cttatacatt aggacctttg cagcataaat 480tactatactt ctatagacac acaaacacaa atacacacac taaattaata 53047867DNAArtificial SequenceSynthetic pACU6Synthetic pACU6 47ttacattatc aatccttgcg tttcagcttc cactaattta gatgactatt tctcatcatt 60tgcgtcatct tctaacaccg tatatgataa tatactagta acgtaaatac tagttagtag 120atgatagttg atttttattc caacactaag aaataatttc gccatttctt gaatgtattt 180aaagatattt aatgctataa tagacattta aatccaattc ttccaacata caatgggagt 240ttggccgagt ggtttaaggc gtcagattta ggtggattta acctctaaaa tctctgatat 300cttcggatgc aagggttcga atcccttagc tctcattatt ttttgctttt tctcttgaat 360tcgaaaaaga catttttgct gtcagtcact gtcaagagat tcttttgctg gcatttcttc 420cagaagcaaa aagagcgatg cgtcttttcc gctgaaccgt tccagcaaaa aagactacca 480acgaattcga aaaagacatt tttgctgtca gtcactgtca agagattctt ttgctggcat 540ttcttccaga agcaaaaaga gcgatgcgtc ttttccgctg aaccgttcca gcaaaaaaga 600ctaccaacga attctaatta agttagtcaa ggcgccatcc tcatgaaaac tgtgtaacat 660aataaccgaa gtgtcgaaaa ggtggcacct tgtccaattg aacacgctcg atgaaaaaaa 720taagatatat ataaggttaa gtaaagcgtc tgttagaaag gaagtttttc ctttttcttg 780ctctcttgtc ttttcatcta ctatttcctt cgtgtaatac agggtcgtca gatacataga 840tacaattcta ttacccccat ccataca 86748867DNAArtificial SequenceSynthetic pACU7Synthetic pACU7 48ttacattatc aatccttgcg tttcagcttc cactaattta gatgactatt tctcatcatt 60tgcgtcatct tctaacaccg tatatgataa tatactagta acgtaaatac tagttagtag 120atgatagttg atttttattc caacactaag aaataatttc gccatttctt gaatgtattt 180aaagatattt aatgctataa tagacattta aatccaattc ttccaacata caatgggagt 240ttggccgagt ggtttaaggc gtcagattta ggtggattta acctctaaaa tctctgatat 300cttcggatgc aagggttcga atcccttagc tctcattatt ttttgctttt tctcttgaat 360tcgttggtag tcttttttgc tggaacggtt cagcggaaaa gacgcatcgc tctttttgct 420tctggaagaa atgccagcaa aagaatctct tgacagtgac tgacagcaaa aatgtctttt 480tcgaattcgt tggtagtctt ttttgctgga acggttcagc ggaaaagacg catcgctctt 540tttgcttctg gaagaaatgc cagcaaaaga atctcttgac agtgactgac agcaaaaatg 600tctttttcga attctaatta agttagtcaa ggcgccatcc tcatgaaaac tgtgtaacat 660aataaccgaa gtgtcgaaaa ggtggcacct tgtccaattg aacacgctcg atgaaaaaaa 720taagatatat ataaggttaa gtaaagcgtc tgttagaaag gaagtttttc ctttttcttg 780ctctcttgtc ttttcatcta ctatttcctt cgtgtaatac agggtcgtca gatacataga 840tacaattcta ttacccccat ccataca 867491119DNAArtificial SequenceSynthetic pACU8Synthetic pACU8 49ttacattatc aatccttgcg tttcagcttc cactaattta gatgactatt tctcatcatt 60tgcgtcatct tctaacaccg tatatgataa tatactagta acgtaaatac tagttagtag 120atgatagttg atttttattc caacactaag aaataatttc gccatttctt gaatgtattt 180aaagatattt aatgctataa tagacattta aatccaattc ttccaacata caatgggagt 240ttggccgagt ggtttaaggc gtcagattta ggtggattta acctctaaaa tctctgatat 300cttcggatgc aagggttcga atcccttagc tctcattatt ttttgctttt tctcttgaat 360tcgaaaaaga catttttgct gtcagtcact gtcaagagat tcttttgctg gcatttcttc 420cagaagcaaa aagagcgatg cgtcttttcc gctgaaccgt tccagcaaaa aagactacca 480acgaattcga aaaagacatt tttgctgtca gtcactgtca agagattctt ttgctggcat 540ttcttccaga agcaaaaaga gcgatgcgtc ttttccgctg aaccgttcca gcaaaaaaga 600ctaccaacga attcgaaaaa gacatttttg ctgtcagtca ctgtcaagag attcttttgc 660tggcatttct tccagaagca aaaagagcga tgcgtctttt ccgctgaacc gttccagcaa 720aaaagactac caacgaattc gaaaaagaca tttttgctgt cagtcactgt caagagattc 780ttttgctggc atttcttcca gaagcaaaaa gagcgatgcg tcttttccgc tgaaccgttc 840cagcaaaaaa gactaccaac gaattctaat taagttagtc aaggcgccat cctcatgaaa 900actgtgtaac ataataaccg aagtgtcgaa aaggtggcac cttgtccaat tgaacacgct 960cgatgaaaaa aataagatat atataaggtt aagtaaagcg tctgttagaa aggaagtttt 1020tcctttttct tgctctcttg tcttttcatc tactatttcc ttcgtgtaat acagggtcgt 1080cagatacata gatacaattc tattaccccc atccataca 111950624DNAArtificial SequenceSynthetic pACU9Synthetic pACU9 50tatagttttt tctccttgac gttaaagtat agaggtatat taacaatttt ttgttgatac 60ttttatgaca tttgaataag aagtaataca aactgaaaat gttgaaagta ttagttaaag 120tggttatgca gcttttccat ttatatatct gttaatagat caaaaatcat cgcttcgctg 180attaattacc ccagaaataa ggctaaaaaa ctaatcgcat tatcatccga attcgaaaaa 240gacatttttg ctgtcagtca ctgtcaagag attcttttgc tggcatttct tccagaagca 300aaaagagcga tgcgtctttt ccgctgaacc gttccagcaa aaaagactac caacgaattc 360gaaaaagaca tttttgctgt cagtcactgt caagagattc

ttttgctggc atttcttcca 420gaagcaaaaa gagcgatgcg tcttttccgc tgaaccgttc cagcaaaaaa gactaccaac 480gaattcgggc tctttacatt tccacaacat ataagtaaga ttagatatgg atatgtatat 540ggtggtaatg ccatgtaata tgattattaa acttctttgc gtccatccaa aaaaaaagta 600agaatttttg aaaattcaat ataa 62451876DNAArtificial SequenceSynthetic pACU10pSynthetic pACU10p 51ttatattgaa ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag aagtttaata 60atcatattac atggcattac caccatatac atatccatat ctaatcttac ttatatgttg 120tggaaatgta aagagcccga attggttggt agtctttttt gctggaacgg ttcagcggaa 180aagacgcatc gctctttttg cttctggaag aaatgccagc aaaagaatct cttgacagtg 240actgacagca aaaatgtctt tttcgaattc gttggtagtc ttttttgctg gaacggttca 300gcggaaaaga cgcatcgctc tttttgcttc tggaagaaat gccagcaaaa gaatctcttg 360acagtgactg acagcaaaaa tgtctttttc gaattcgttg gtagtctttt ttgctggaac 420ggttcagcgg aaaagacgca tcgctctttt tgcttctgga agaaatgcca gcaaaagaat 480ctcttgacag tgactgacag caaaaatgtc tttttcgaat tcgttggtag tcttttttgc 540tggaacggtt cagcggaaaa gacgcatcgc tctttttgct tctggaagaa atgccagcaa 600aagaatctct tgacagtgac tgacagcaaa aatgtctttt tccaattcgg atgataatgc 660gattagtttt ttagccttat ttctggggta attaatcagc gaagcgatga tttttgatct 720attaacagat atataaatgg aaaagctgca taaccacttt aactaatact ttcaacattt 780tcagtttgta ttacttctta ttcaaatgtc ataaaagtat caacaaaaaa ttgttaatat 840acctctatac tttaacgtca aggagaaaaa actata 87652633DNAArtificial SequenceSynthetic pACU11Synthetic pACU11 52gctcagcatc tgcttcttcc caaagatgaa cgcggcgtta tgtcactaac gacgtgcacc 60aacttgcggg aattcgaaaa agacattttt gctgtcagtc actgtcaaga gattcttttg 120ctggcatttc ttccagaagc aaaaagagcg atgcgtcttt tccgctgaac cgttccagca 180aaaaagacta ccaacgaatt ccaccgcacg ccttttttct gaagcccact ttcgtggact 240ttgccatata tgcaaaattc atgaagtgtg ataccaagtc agcatacacc tcactagggt 300agtttctttg gttgtattga tcatttggtt catcgtggtt cattaatttt ttttctccat 360tgctttctgg ctttgatctt actatcattt ggatttttgt cgaaggttgt agaattgtat 420gtgacaagtg gcaccaagca tatataaaaa aaaaaagcat tatcttccta ccagagttga 480ttgttaaaaa cgtatttata gcaaacgcaa ttgtaattaa ttcttatttt gtatcttttc 540ttcccttgtc tcaatctttt atttttattt tatttttctt ttcttagttt ctttcataac 600accaagcaac taatactata acatacaata ata 633531119DNAArtificial SequenceSynthetic pACU12Synthetic pACU12 53ttacattatc aatccttgcg tttcagcttc cactaattta gatgactatt tctcatcatt 60tgcgtcatct tctaacaccg tatatgataa tatactagta acgtaaatac tagttagtag 120atgatagttg atttttattc caacactaag aaataatttc gccatttctt gaatgtattt 180aaagatattt aatgctataa tagacattta aatccaattc ttccaacata caatgggagt 240ttggccgagt ggtttaaggc gtcagattta ggtggattta acctctaaaa tctctgatat 300cttcggatgc aagggttcga atcccttagc tctcattatt ttttgctttt tctcttgaat 360tggttggtag tcttttttgc tggaacggtt cagcggaaaa gacgcatcgc tctttttgct 420tctggaagaa atgccagcaa aagaatctct tgacagtgac tgacagcaaa aatgtctttt 480tcgaattcgt tggtagtctt ttttgctgga acggttcagc ggaaaagacg catcgctctt 540tttgcttctg gaagaaatgc cagcaaaaga atctcttgac agtgactgac agcaaaaatg 600tctttttcga attcgttggt agtctttttt gctggaacgg ttcagcggaa aagacgcatc 660gctctttttg cttctggaag aaatgccagc aaaagaatct cttgacagtg actgacagca 720aaaatgtctt tttcgaattc gttggtagtc ttttttgctg gaacggttca gcggaaaaga 780cgcatcgctc tttttgcttc tggaagaaat gccagcaaaa gaatctcttg acagtgactg 840acagcaaaaa tgtctttttc caattctaat taagttagtc aaggcgccat cctcatgaaa 900actgtgtaac ataataaccg aagtgtcgaa aaggtggcac cttgtccaat tgaacacgct 960cgatgaaaaa aataagatat atataaggtt aagtaaagcg tctgttagaa aggaagtttt 1020tcctttttct tgctctcttg tcttttcatc tactatttcc ttcgtgtaat acagggtcgt 1080cagatacata gatacaattc tattaccccc atccataca 1119541497DNAArtificial SequenceSynthetic pACU13Synthetic pACU13 54ttacattatc aatccttgcg tttcagcttc cactaattta gatgactatt tctcatcatt 60tgcgtcatct tctaacaccg tatatgataa tatactagta acgtaaatac tagttagtag 120atgatagttg atttttattc caacactaag aaataatttc gccatttctt gaatgtattt 180aaagatattt aatgctataa tagacattta aatccaattc ttccaacata caatgggagt 240ttggccgagt ggtttaaggc gtcagattta ggtggattta acctctaaaa tctctgatat 300cttcggatgc aagggttcga atcccttagc tctcattatt ttttgctttt tctcttgaat 360tggttggtag tcttttttgc tggaacggtt cagcggaaaa gacgcatcgc tctttttgct 420tctggaagaa atgccagcaa aagaatctct tgacagtgac tgacagcaaa aatgtctttt 480tcgaattcgt tggtagtctt ttttgctgga acggttcagc ggaaaagacg catcgctctt 540tttgcttctg gaagaaatgc cagcaaaaga atctcttgac agtgactgac agcaaaaatg 600tctttttcga attcgttggt agtctttttt gctggaacgg ttcagcggaa aagacgcatc 660gctctttttg cttctggaag aaatgccagc aaaagaatct cttgacagtg actgacagca 720aaaatgtctt tttcgaattc gttggtagtc ttttttgctg gaacggttca gcggaaaaga 780cgcatcgctc tttttgcttc tggaagaaat gccagcaaaa gaatctcttg acagtgactg 840acagcaaaaa tgtctttttc gaattcgttg gtagtctttt ttgctggaac ggttcagcgg 900aaaagacgca tcgctctttt tgcttctgga agaaatgcca gcaaaagaat ctcttgacag 960tgactgacag caaaaatgtc tttttcgaat tcgttggtag tcttttttgc tggaacggtt 1020cagcggaaaa gacgcatcgc tctttttgct tctggaagaa atgccagcaa aagaatctct 1080tgacagtgac tgacagcaaa aatgtctttt tcgaattcgt tggtagtctt ttttgctgga 1140acggttcagc ggaaaagacg catcgctctt tttgcttctg gaagaaatgc cagcaaaaga 1200atctcttgac agtgactgac agcaaaaatg tctttttcca attctaatta agttagtcaa 1260ggcgccatcc tcatgaaaac tgtgtaacat aataaccgaa gtgtcgaaaa ggtggcacct 1320tgtccaattg aacacgctcg atgaaaaaaa taagatatat ataaggttaa gtaaagcgtc 1380tgttagaaag gaagtttttc ctttttcttg ctctcttgtc ttttcatcta ctatttcctt 1440cgtgtaatac agggtcgtca gatacataga tacaattcta ttacccccat ccataca 1497551011DNAArtificial SequenceSynthetic pACU14Synthetic pACU14 55gctcagcatc tgcttcttcc caaagatgaa cgcggcgtta tgtcactaac gacgtgcacc 60aacttgcggg aattggaaaa agacattttt gctgtcagtc actgtcaaga gattcttttg 120ctggcatttc ttccagaagc aaaaagagcg atgcgtcttt tccgctgaac cgttccagca 180aaaaagacta ccaacgaatt cgaaaaagac atttttgctg tcagtcactg tcaagagatt 240cttttgctgg catttcttcc agaagcaaaa agagcgatgc gtcttttccg ctgaaccgtt 300ccagcaaaaa agactaccaa cgaattcgaa aaagacattt ttgctgtcag tcactgtcaa 360gagattcttt tgctggcatt tcttccagaa gcaaaaagag cgatgcgtct tttccgctga 420accgttccag caaaaaagac taccaacgaa ttcgaaaaag acatttttgc tgtcagtcac 480tgtcaagaga ttcttttgct ggcatttctt ccagaagcaa aaagagcgat gcgtcttttc 540cgctgaaccg ttccagcaaa aaagactacc aaccaattcc accgcacgcc ttttttctga 600agcccacttt cgtggacttt gccatatatg caaaattcat gaagtgtgat accaagtcag 660catacacctc actagggtag tttctttggt tgtattgatc atttggttca tcgtggttca 720ttaatttttt ttctccattg ctttctggct ttgatcttac tatcatttgg atttttgtcg 780aaggttgtag aattgtatgt gacaagtggc accaagcata tataaaaaaa aaaagcatta 840tcttcctacc agagttgatt gttaaaaacg tatttatagc aaacgcaatt gtaattaatt 900cttattttgt atcttttctt cccttgtctc aatcttttat ttttatttta tttttctttt 960cttagtttct ttcataacac caagcaacta atactataac atacaataat a 101156398DNAArtificial SequenceSynthetic pACU15Synthetic pACU15 56tatagttttt tctccttgac gttaaagtat agaggtatat taacaatttt ttgttgatac 60ttttatgaca tttgaataag aagtaataca aactgaaaat gttgaaagta ttagttaaag 120tggttatgca gcttttccat ttatatatct gttaatagat caaaaatcat cgcttcgctg 180attaattacc ccagaaataa ggctaaaaaa ctaatcgcat tatcatccga attcgttggt 240agtctttttt gctggaacgg ttcagcggaa aagacgcatc gctctttttg cttctggaag 300aaatgccagc aaaagaatct cttgacagtg actgacagca aaaatgtctt tttcgaattc 360gggctcttta catttccaca acatataagt aagattag 39857428DNAArtificial SequenceSynthetic pGAL/CUP1pSynthetic pGAL/CUP1p 57ttatattgaa ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag aagtttaata 60atcatattac atggcattac caccatatac atatccatat ctaatcttac ttatatgttg 120tggaaatgta aagagcccga attcgaaaaa gacatttttg ctgtcagtca ctgtcaagag 180attcttttgc tggcatttct tccagaagca aaaagagcga tgcgtctttt ccgctgaacc 240gttccagcaa aaaagactac caacgcaata tggattgtca gaatcatata aaagagaagc 300aaataactcc ttgtcttgta tcaattgcat tataatatct tcttgttagt gcaatatcat 360atagaagtca tcgaaataga tattaagaaa aacaaactgt acaatcaatc aatcaatcat 420cacataaa 42858518DNASaccharomyces cerevisiaepCRS5pCRS5 58gtggacgaaa agacataact gcagaagtac agctgccttt atttcttgtg gtcatttatt 60gcttttattt tcaagtcaga tatacaagaa aatcaaatcc catcgtcaac gtcacgtata 120aacgattaat ttacagtaat accatactct accaacatta ttttagtccg acgttcagtc 180ctgtaggtgt tccaaatcct tctggcattg acttctgtgc agaaaccctt caaaatgagt 240tccactttac gtcagatcgc ataacaaccg gtcatatatt tttttctttt gctaaacccc 300ctactgcaag cacttttaag aaaaagaaca ataaatgcgt ctttattgct gtgtggaagt 360gatttttgtc tttcggacaa aaaaaggata gggatgcgag agggctgtga agtagtgatc 420aagcggggcc tatataagaa gggcgcacat cgtcccccct aagaatagcg aagcgatatt 480acactgaaca ctacaatgtc aaatagtact caataaat 51859510DNASaccharomyces cerevisiaepCHA1pCHA1 59gatctctgct gacgttgtat ccacagatct aattgcaaga tagcctcttg cgaccttatt 60aaaagcctct ccgtgatatc ctctagggct tgggttgcca ttaatcgatg tgtccttgtt 120tccttatgcg agctgtttct tatctatctt atggtcccat tctttactgc actgtttaca 180ttttgatcaa ttgcgaaatg ttcctactat ttttcttttt ctcttttcgc gagtactaat 240caccgcgaac ggaaactaat gagtcctctg cgcggagaca tgattccgca tgggcggctc 300ctgttaagcc ccagcggaaa tgtaattcca ctgagtgtca ttaaatagtg ccaaagcttt 360atcaaattgt ttgcgatgag ataagataaa agggacaata tgaggaggaa cacaggtata 420taaatatcgc caaataaaag gaaaatgttt atacagtttt ctctttttta agtgctggat 480agacaagaga caggaaaatt aaccagcgag 51060601DNASaccharomyces cerevisiaepCTR1pCTR1 60caagtccgat tgttcctctt caggagcttc ctgaaccaaa ctttttccgc aaggccgcat 60tttgaaccgt attttgctcg ttccagcctt tccacgtttt tgttatctaa gcaacttggc 120acatttccct actatactac aaaccgatac gtaaatactt ccctaaatag catatgaatt 180attcagtaat ttttaaggat cgaaactgca cctcaactat tcgttactgt ggttatgttc 240tcatgtattg atgcaaatca tgggatattt gctcaagacg acggtaaaat gagcaaaaat 300ggcacgatcc tgaaaagagc acttttcaag attcgggcta caaaatgcaa cataaaaaat 360gttgtattgt catctcgaca gggtcttgta tgttttattc ctcttatgat tagttcacat 420tagtaaaaca gatacgcagt gtgctcttaa taaacaacta ctccatagct ttatttgcat 480aacaaaactt ttaagcacaa acttaaacag gtggagtaat agttcggcgg cgactcaaat 540tacatttgtt ggaagaatcg aatagaaaat aaaaaaaagt gtattatatt tgacattcaa 600a 60161522DNASaccharomyces cerevisiaepCTR3pCTR3 61gatgtgatga caaaacctct tccgataaaa acatttaaac tattaacaaa caaatggatt 60cattagatct attacattat gggtggtatg ttggaataaa aatcaactat catctactaa 120ctagtattta cgttactagt atattatcat atacggtgtt agaagatgac gcaaatgatg 180agaaatagtc atctaaatta gtggaagctg aaacgcaagg attgataatg taataggatc 240aatgaatatt aacatataaa acgatgataa taatatttat agaattgtgt agaattgcag 300attccctttt atggattcct aaatcctcca ggagaacttc tagtatatct acatacctaa 360tattattgcc ttattaaaaa tggaatccca acaattacat caaaatccac attctcttca 420cttctccgat agacttgtaa tttatcttat ttcatttcct aacactttga tcgaagaaga 480gggataacaa cagacgaaaa cacatttaag ggctatacaa ag 52262675DNAArtificial SequenceSynthetic pCUR1Synthetic pCUR1 62ttacattatc aatccttgcg tttcagcttc cactaattta gatgactatt tctcatcatt 60tgcgtcatct tctaacaccg tatatgataa tatactagta acgtaaatac tagttagtag 120atgatagttg atttttattc caacactaag aaataatttc gccatttctt gaatgtattt 180aaagatattt aatgctataa tagacattta aatccaattc ttccaacata caatgggagt 240ttggccgagt ggtttaaggc gtcagattta ggtggattta acctctaaaa tctctgatat 300cttcggatgc aagggttcga atcccttagc tctcattatt ttttgctttt tctcttgaat 360tgtcatggga tatttgctca agacgacggt aaaatgagca aatatggcac gatcctcaat 420tctaattaag ttagtcaagg cgccatcctc atgaaaactg tgtaacataa taaccgaagt 480gtcgaaaagg tggcaccttg tccaattgaa cacgctcgat gaaaaaaata agatatatat 540aaggttaagt aaagcgtctg ttagaaagga agtttttcct ttttcttgct ctcttgtctt 600ttcatctact atttccttcg tgtaatacag ggtcgtcaga tacatagata caattctatt 660acccccatcc ataca 67563674DNAArtificial SequenceSynthetic pCUR2Synthetic pCUR2 63ttacattatc aatccttgcg tttcagcttc cactaattta gatgactatt tctcatcatt 60tgcgtcatct tctaacaccg tatatgataa tatactagta acgtaaatac tagttagtag 120atgatagttg atttttattc caacactaag aaataatttc gccatttctt gaatgtattt 180aaagatattt aatgctataa tagacattta aatccaattc ttccaacata caatgggagt 240ttggccgagt ggtttaaggc gtcagattta ggtggattta acctctaaaa tctctgatat 300cttcggatgc aagggttcga atcccttagc tctcattatt ttttgctttt tctcttgatt 360gaggatcgtg ccatatttgc tcattttacc gtcgtcttga gcaaatatcc catgacaatt 420ctaattaagt tagtcaaggc gccatcctca tgaaaactgt gtaacataat aaccgaagtg 480tcgaaaaggt ggcaccttgt ccaattgaac acgctcgatg aaaaaaataa gatatatata 540aggttaagta aagcgtctgt tagaaaggaa gtttttcctt tttcttgctc tcttgtcttt 600tcatctacta tttccttcgt gtaatacagg gtcgtcagat acatagatac aattctatta 660cccccatcca taca 67464794DNAArtificial SequenceSynthetic pCUR3Synthetic pCUR3 64ttacattatc aatccttgcg tttcagcttc cactaattta gatgactatt tctcatcatt 60tgcgtcatct tctaacaccg tatatgataa tatactagta acgtaaatac tagttagtag 120atgatagttg atttttattc caacactaag aaataatttc gccatttctt gaatgtattt 180aaagatattt aatgctataa tagacattta aatccaattc ttccaacata caatgggagt 240ttggccgagt ggtttaaggc gtcagattta ggtggattta acctctaaaa tctctgatat 300cttcggatgc aagggttcga atcccttagc tctcattatt ttttgctttt tctcttgaat 360taggatcgtg ccatatttgc tcattttacc gtcgtcttga gcaaatatcc catgacaatt 420gaggatcgtg ccatatttgc tcattttacc gtcgtcttga gcaaatatcc catgacaatt 480gaggatcgtg ccatatttgc tcattttacc gtcgtcttga gcaaatatcc catgacaatt 540ctaattaagt tagtcaaggc gccatcctca tgaaaactgt gtaacataat aaccgaagtg 600tcgaaaaggt ggcaccttgt ccaattgaac acgctcgatg aaaaaaataa gatatatata 660aggttaagta aagcgtctgt tagaaaggaa gtttttcctt tttcttgctc tcttgtcttt 720tcatctacta tttccttcgt gtaatacagg gtcgtcagat acatagatac aattctatta 780cccccatcca taca 79465850DNAArtificial SequenceSynthetic pCUR4Synthetic pCUR4 65ttacattatc aatccttgcg tttcagcttc cactaattta gatgactatt tctcatcatt 60tgcgtcatct tctaacaccg tatatgataa tatactagta acgtaaatac tagttagtag 120atgatagttg atttttattc caacactaag aaataatttc gccatttctt gaatgtattt 180aaagatattt aatgctataa tagacattta aatccaattc ttccaacata caatgggagt 240ttggccgagt ggtttaaggc gtcagattta ggtggattta acctctaaaa tctctgatat 300cttcggatgc aagggttcga atcccttagc tctcattatt ttttgctttt tctcttgaat 360tgtcatggga tatttgctca agacgacggt aaaatgagca aatatggcac gatcctcaat 420tgtcatggga tatttgctca agacgacggt aaaatgagca aatatggcac gatcctcaat 480gtcatgggat atttgctcaa gacgacggta aaatgagcaa atatggcacg atcctcaatt 540gtcatgggat atttgctcaa gacgacggta aaatgagcaa atatcccatg acaattctaa 600ttaagttagt caaggcgcca tcctcatgaa aactgtgtaa cataataacc gaagtgtcga 660aaaggtggca ccttgtccaa ttgaacacgc tcgatgaaaa aaataagata tatataaggt 720taagtaaagc gtctgttaga aaggaagttt ttcctttttc ttgctctctt gtcttttcat 780ctactatttc cttcgtgtaa tacagggtcg tcagatacat agatacaatt ctattacccc 840catccataca 85066491DNAArtificial SequenceSynthetic pCUR5pSynthetic pCUR5p 66ttatattgaa ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag aagtttaata 60atcatattac atggcattac caccatatac atatccatat ctaatcttac ttatatgttg 120tggaaatgta aagagcccga attgtcatgg gatatttgct caagacgacg gtaaaatgag 180caaatatggc acgatcctca attgtcatgg gatatttgct caagacgacg gtaaaatgag 240caaatatggc acgatcccaa ttcggatgat aatgcgatta gttttttagc cttatttctg 300gggtaattaa tcagcgaagc gatgattttt gatctattaa cagatatata aatggaaaag 360ctgcataacc actttaacta atactttcaa cattttcagt ttgtattact tcttattcaa 420atgtcataaa agtatcaaca aaaaattgtt aatatacctc tatactttaa cgtcaaggag 480aaaaaactat a 49167833DNAArtificial SequenceSynthetic pCUR6Synthetic pCUR6 67ttacattatc aatccttgcg tttcagcttc cactaattta gatgactatt tctcatcatt 60tgcgtcatct tctaacaccg tatatgataa tatactagta acgtaaatac tagttagtag 120atgatagttg atttttattc caacactaag aaataatttc gccatttctt gaatgtattt 180aaagatattt aatgctataa tagacattta aatccaattc ttccaacata caatgggagt 240ttggccgagt ggtttaaggc gtcagattta ggtggattta acctctaaaa tctctgatat 300cttcggatgc aagggttcga atcccgaatt gaggatcgtg ccatatttgc tcattttacc 360gtcgtcttga gcaaatatcc catgacaatt gaggatcgtg ccatatttgc tcattttacc 420gtcgtcttga gcaaatatcc catgacaatt gaggatcgtg ccatatttgc tcattttacc 480gtcgtcttga gcaaatatcc catgacaatt catgatcgca aaatggcaaa tggcacgtga 540agctgtcgat attggggaac tgtggtggtt ggcaaatgac taattaagtt agtcaaggcg 600ccatcctcat gaaaactgtg taacataata accgaagtgt cgaaaaggtg gcaccttgtc 660caattgaaca cgctcgatga aaaaaataag atatatataa ggttaagtaa agcgtctgtt 720agaaaggaag tttttccttt ttcttgctct cttgtctttt catctactat ttccttcgtg 780taatacaggg tcgtcagata catagataca attctattac ccccatccat aca 83368803DNAArtificial SequenceSynthetic pCUR7Synthetic pCUR7 68gtgagtaagg aaagagtgag gaactatcgc atacctgcat ttaaagatgc cgatttgggc 60gcgaatcctt tattttggct tcaccctcat actattatca gggccagaaa aaggaagtgt 120ttccctcctt cttgaattga tgttaccctc ataaagcacg tggcctctta tcgagaaaga 180aattaccgtc gctcgtgatt tgtttgcaaa aagaacaaaa ctgaattcag gatcgtgcca 240tatttgctca ttttaccgtc gtcttgagca aatatcccat gacaattgag gatcgtgcca 300tatttgctca ttttaccgtc gtcttgagca aatatcccat gagaattctt cctgtcttcc 360tattgattgc agcttccaat ttcgtcacac aacaaggtcc tagcgacggc tcacaggttt 420tgtaacaagc aatcgaaggt tctggaatgg cgggaaaggg tttagtacca catgctatga 480tgcccactgt gatctccaga gcaaagttcg ttcgatcgta ctgttactct ctctctttca 540aacagaattg tccgaatcgt gtgacaacaa cagcctgttc tcacacactc ttttcttcta 600accaaggggg tggtttagtt tagtagaacc tcgtgaaact tacatttaca tatatataaa 660cttgcataaa ttggtcaatg caagaaatac atatttggtc ttttctaatt cgtagttttt 720caagttctta gatgctttct ttttctcttt tttacagatc atcaaggaag taattatcta 780ctttttacaa caaatataaa aca

80369863DNAArtificial SequenceSynthetic pCUR8Synthetic pCUR8 69gtgagtaagg aaagagtgag gaactatcgc atacctgcat ttaaagatgc cgatttgggc 60gcgaatcctt tattttggct tcaccctcat actattatca gggccagaaa aaggaagtgt 120ttccctcctt cttgaattga tgttaccctc ataaagcacg tggcctctta tcgagaaaga 180aattaccgtc gctcgtgatt tgtttgcaaa aagaacaaaa ctgaattcag gatcgtgcca 240tatttgctca ttttaccgtc gtcttgagca aatatcccat gacaattgag gatcgtgcca 300tatttgctca ttttaccgtc gtcttgagca aatatcccat gacaattgag gatcgtgcca 360tatttgctca ttttaccgtc gtcttgagca aatatcccat gagaattctt cctgtcttcc 420tattgattgc agcttccaat ttcgtcacac aacaaggtcc tagcgacggc tcacaggttt 480tgtaacaagc aatcgaaggt tctggaatgg cgggaaaggg tttagtacca catgctatga 540tgcccactgt gatctccaga gcaaagttcg ttcgatcgta ctgttactct ctctctttca 600aacagaattg tccgaatcgt gtgacaacaa cagcctgttc tcacacactc ttttcttcta 660accaaggggg tggtttagtt tagtagaacc tcgtgaaact tacatttaca tatatataaa 720cttgcataaa ttggtcaatg caagaaatac atatttggtc ttttctaatt cgtagttttt 780caagttctta gatgctttct ttttctcttt tttacagatc atcaaggaag taattatcta 840ctttttacaa caaatataaa aca 86370863DNAArtificial SequenceSynthetic pCUR9Synthetic pCUR9 70gtgagtaagg aaagagtgag gaactatcgc atacctgcat ttaaagatgc cgatttgggc 60gcgaatcctt tattttggct tcaccctcat actattatca gggccagaaa aaggaagtgt 120ttccctcctt cttgaattga tgttaccctc ataaagcacg tggcctctta tcgagaaaga 180aattaccgtc gctcgtgatt tgtttgcaaa aagaacaaaa ctgaattctc atgggatatt 240tgctcaagac gacggtaaaa tgagcaaata tggcacgatc ctcaattgtc atgggatatt 300tgctcaagac gacggtaaaa tgagcaaata tggcacgatc ctcaattgtc atgggatatt 360tgctcaagac gacggtaaaa tgagcaaata tggcacgatc ctgaattctt cctgtcttcc 420tattgattgc agcttccaat ttcgtcacac aacaaggtcc tagcgacggc tcacaggttt 480tgtaacaagc aatcgaaggt tctggaatgg cgggaaaggg tttagtacca catgctatga 540tgcccactgt gatctccaga gcaaagttcg ttcgatcgta ctgttactct ctctctttca 600aacagaattg tccgaatcgt gtgacaacaa cagcctgttc tcacacactc ttttcttcta 660accaaggggg tggtttagtt tagtagaacc tcgtgaaact tacatttaca tatatataaa 720cttgcataaa ttggtcaatg caagaaatac atatttggtc ttttctaatt cgtagttttt 780caagttctta gatgctttct ttttctcttt tttacagatc atcaaggaag taattatcta 840ctttttacaa caaatataaa aca 86371923DNAArtificial SequenceSynthetic pCUR10Synthetic pCUR10 71gtgagtaagg aaagagtgag gaactatcgc atacctgcat ttaaagatgc cgatttgggc 60gcgaatcctt tattttggct tcaccctcat actattatca gggccagaaa aaggaagtgt 120ttccctcctt cttgaattga tgttaccctc ataaagcacg tggcctctta tcgagaaaga 180aattaccgtc gctcgtgatt tgtttgcaaa aagaacaaaa ctgaattctc atgggatatt 240tgctcaagac gacggtaaaa tgagcaaata tggcacgatc ctcaattgtc atgggatatt 300tgctcaagac gacggtaaaa tgagcaaata tggcacgatc ctcaattgtc atgggatatt 360tgctcaagac gacggtaaaa tgagcaaata tggcacgatc ctcaattgtc atgggatatt 420tgctcaagac gacggtaaaa tgagcaaata tggcacgatc ctgaattctt cctgtcttcc 480tattgattgc agcttccaat ttcgtcacac aacaaggtcc tagcgacggc tcacaggttt 540tgtaacaagc aatcgaaggt tctggaatgg cgggaaaggg tttagtacca catgctatga 600tgcccactgt gatctccaga gcaaagttcg ttcgatcgta ctgttactct ctctctttca 660aacagaattg tccgaatcgt gtgacaacaa cagcctgttc tcacacactc ttttcttcta 720accaaggggg tggtttagtt tagtagaacc tcgtgaaact tacatttaca tatatataaa 780cttgcataaa ttggtcaatg caagaaatac atatttggtc ttttctaatt cgtagttttt 840caagttctta gatgctttct ttttctcttt tttacagatc atcaaggaag taattatcta 900ctttttacaa caaatataaa aca 92372983DNAArtificial SequenceSynthetic pCUR11Synthetic pCUR11 72gtgagtaagg aaagagtgag gaactatcgc atacctgcat ttaaagatgc cgatttgggc 60gcgaatcctt tattttggct tcaccctcat actattatca gggccagaaa aaggaagtgt 120ttccctcctt cttgaattga tgttaccctc ataaagcacg tggcctctta tcgagaaaga 180aattaccgtc gctcgtgatt tgtttgcaaa aagaacaaaa ctgaattctc atgggatatt 240tgctcaagac gacggtaaaa tgagcaaata tggcacgatc ctcaattgtc atgggatatt 300tgctcaagac gacggtaaaa tgagcaaata tggcacgatc ctcaattgtc atgggatatt 360tgctcaagac gacggtaaaa tgagcaaata tggcacgatc ctcaattgtc atgggatatt 420tgctcaagac gacggtaaaa tgagcaaata tggcacgatc ctcaattgtc atgggatatt 480tgctcaagac gacggtaaaa tgagcaaata tggcacgatc ctgaattctt cctgtcttcc 540tattgattgc agcttccaat ttcgtcacac aacaaggtcc tagcgacggc tcacaggttt 600tgtaacaagc aatcgaaggt tctggaatgg cgggaaaggg tttagtacca catgctatga 660tgcccactgt gatctccaga gcaaagttcg ttcgatcgta ctgttactct ctctctttca 720aacagaattg tccgaatcgt gtgacaacaa cagcctgttc tcacacactc ttttcttcta 780accaaggggg tggtttagtt tagtagaacc tcgtgaaact tacatttaca tatatataaa 840cttgcataaa ttggtcaatg caagaaatac atatttggtc ttttctaatt cgtagttttt 900caagttctta gatgctttct ttttctcttt tttacagatc atcaaggaag taattatcta 960ctttttacaa caaatataaa aca 983731043DNAArtificial SequenceSynthetic pCUR12Synthetic pCUR12 73gtgagtaagg aaagagtgag gaactatcgc atacctgcat ttaaagatgc cgatttgggc 60gcgaatcctt tattttggct tcaccctcat actattatca gggccagaaa aaggaagtgt 120ttccctcctt cttgaattga tgttaccctc ataaagcacg tggcctctta tcgagaaaga 180aattaccgtc gctcgtgatt tgtttgcaaa aagaacaaaa ctgaattcag gatcgtgcca 240tatttgctca ttttaccgtc gtcttgagca aatatcccat gacaattgag gatcgtgcca 300tatttgctca ttttaccgtc gtcttgagca aatatcccat gacaattgag gatcgtgcca 360tatttgctca ttttaccgtc gtcttgagca aatatcccat gagaattcag gatcgtgcca 420tatttgctca ttttaccgtc gtcttgagca aatatcccat gacaattgag gatcgtgcca 480tatttgctca ttttaccgtc gtcttgagca aatatcccat gacaattgag gatcgtgcca 540tatttgctca ttttaccgtc gtcttgagca aatatcccat gagaattctt cctgtcttcc 600tattgattgc agcttccaat ttcgtcacac aacaaggtcc tagcgacggc tcacaggttt 660tgtaacaagc aatcgaaggt tctggaatgg cgggaaaggg tttagtacca catgctatga 720tgcccactgt gatctccaga gcaaagttcg ttcgatcgta ctgttactct ctctctttca 780aacagaattg tccgaatcgt gtgacaacaa cagcctgttc tcacacactc ttttcttcta 840accaaggggg tggtttagtt tagtagaacc tcgtgaaact tacatttaca tatatataaa 900cttgcataaa ttggtcaatg caagaaatac atatttggtc ttttctaatt cgtagttttt 960caagttctta gatgctttct ttttctcttt tttacagatc atcaaggaag taattatcta 1020ctttttacaa caaatataaa aca 1043741043DNAArtificial SequenceSynthetic pCUR13Synthetic pCUR13 74gtgagtaagg aaagagtgag gaactatcgc atacctgcat ttaaagatgc cgatttgggc 60gcgaatcctt tattttggct tcaccctcat actattatca gggccagaaa aaggaagtgt 120ttccctcctt cttgaattga tgttaccctc ataaagcacg tggcctctta tcgagaaaga 180aattaccgtc gctcgtgatt tgtttgcaaa aagaacaaaa ctgaattctc atgggatatt 240tgctcaagac gacggtaaaa tgagcaaata tggcacgatc ctcaattgtc atgggatatt 300tgctcaagac gacggtaaaa tgagcaaata tggcacgatc ctcaattgtc atgggatatt 360tgctcaagac gacggtaaaa tgagcaaata tggcacgatc ctgaattctc atgggatatt 420tgctcaagac gacggtaaaa tgagcaaata tggcacgatc ctcaattgtc atgggatatt 480tgctcaagac gacggtaaaa tgagcaaata tggcacgatc ctcaattgtc atgggatatt 540tgctcaagac gacggtaaaa tgagcaaata tggcacgatc ctgaattctt cctgtcttcc 600tattgattgc agcttccaat ttcgtcacac aacaaggtcc tagcgacggc tcacaggttt 660tgtaacaagc aatcgaaggt tctggaatgg cgggaaaggg tttagtacca catgctatga 720tgcccactgt gatctccaga gcaaagttcg ttcgatcgta ctgttactct ctctctttca 780aacagaattg tccgaatcgt gtgacaacaa cagcctgttc tcacacactc ttttcttcta 840accaaggggg tggtttagtt tagtagaacc tcgtgaaact tacatttaca tatatataaa 900cttgcataaa ttggtcaatg caagaaatac atatttggtc ttttctaatt cgtagttttt 960caagttctta gatgctttct ttttctcttt tttacagatc atcaaggaag taattatcta 1020ctttttacaa caaatataaa aca 1043751223DNAArtificial SequenceSynthetic pCUR14Synthetic pCUR14 75gtgagtaagg aaagagtgag gaactatcgc atacctgcat ttaaagatgc cgatttgggc 60gcgaatcctt tattttggct tcaccctcat actattatca gggccagaaa aaggaagtgt 120ttccctcctt cttgaattga tgttaccctc ataaagcacg tggcctctta tcgagaaaga 180aattaccgtc gctcgtgatt tgtttgcaaa aagaacaaaa ctgaattctc atgggatatt 240tgctcaagac gacggtaaaa tgagcaaata tggcacgatc ctcaattgtc atgggatatt 300tgctcaagac gacggtaaaa tgagcaaata tggcacgatc ctcaattgtc atgggatatt 360tgctcaagac gacggtaaaa tgagcaaata tggcacgatc ctgaattctc atgggatatt 420tgctcaagac gacggtaaaa tgagcaaata tggcacgatc ctcaattgtc atgggatatt 480tgctcaagac gacggtaaaa tgagcaaata tggcacgatc ctcaattgtc atgggatatt 540tgctcaagac gacggtaaaa tgagcaaata tggcacgatc ctgaattctc atgggatatt 600tgctcaagac gacggtaaaa tgagcaaata tggcacgatc ctcaattgtc atgggatatt 660tgctcaagac gacggtaaaa tgagcaaata tggcacgatc ctcaattgtc atgggatatt 720tgctcaagac gacggtaaaa tgagcaaata tggcacgatc ctgaattctt cctgtcttcc 780tattgattgc agcttccaat ttcgtcacac aacaaggtcc tagcgacggc tcacaggttt 840tgtaacaagc aatcgaaggt tctggaatgg cgggaaaggg tttagtacca catgctatga 900tgcccactgt gatctccaga gcaaagttcg ttcgatcgta ctgttactct ctctctttca 960aacagaattg tccgaatcgt gtgacaacaa cagcctgttc tcacacactc ttttcttcta 1020accaaggggg tggtttagtt tagtagaacc tcgtgaaact tacatttaca tatatataaa 1080cttgcataaa ttggtcaatg caagaaatac atatttggtc ttttctaatt cgtagttttt 1140caagttctta gatgctttct ttttctcttt tttacagatc atcaaggaag taattatcta 1200ctttttacaa caaatataaa aca 1223761283DNAArtificial SequenceSynthetic pCUR15Synthetic pCUR15 76gtgagtaagg aaagagtgag gaactatcgc atacctgcat ttaaagatgc cgatttgggc 60gcgaatcctt tattttggct tcaccctcat actattatca gggccagaaa aaggaagtgt 120ttccctcctt cttgaattga tgttaccctc ataaagcacg tggcctctta tcgagaaaga 180aattaccgtc gctcgtgatt tgtttgcaaa aagaacaaaa ctgaattcag gatcgtgcca 240tatttgctca ttttaccgtc gtcttgagca aatatcccat gacaattgag gatcgtgcca 300tatttgctca ttttaccgtc gtcttgagca aatatcccat gacaattgag gatcgtgcca 360tatttgctca ttttaccgtc gtcttgagca aatatcccat gacaattgag gatcgtgcca 420tatttgctca ttttaccgtc gtcttgagca aatatcccat gagaattcag gatcgtgcca 480tatttgctca ttttaccgtc gtcttgagca aatatcccat gacaattgag gatcgtgcca 540tatttgctca ttttaccgtc gtcttgagca aatatcccat gacaattgag gatcgtgcca 600tatttgctca ttttaccgtc gtcttgagca aatatcccat gagaattcag gatcgtgcca 660tatttgctca ttttaccgtc gtcttgagca aatatcccat gacaattgag gatcgtgcca 720tatttgctca ttttaccgtc gtcttgagca aatatcccat gacaattgag gatcgtgcca 780tatttgctca ttttaccgtc gtcttgagca aatatcccat gagaattctt cctgtcttcc 840tattgattgc agcttccaat ttcgtcacac aacaaggtcc tagcgacggc tcacaggttt 900tgtaacaagc aatcgaaggt tctggaatgg cgggaaaggg tttagtacca catgctatga 960tgcccactgt gatctccaga gcaaagttcg ttcgatcgta ctgttactct ctctctttca 1020aacagaattg tccgaatcgt gtgacaacaa cagcctgttc tcacacactc ttttcttcta 1080accaaggggg tggtttagtt tagtagaacc tcgtgaaact tacatttaca tatatataaa 1140cttgcataaa ttggtcaatg caagaaatac atatttggtc ttttctaatt cgtagttttt 1200caagttctta gatgctttct ttttctcttt tttacagatc atcaaggaag taattatcta 1260ctttttacaa caaatataaa aca 128377686DNAArtificial SequenceSynthetic pCUR16Synthetic pCUR16 77gctcagcatc tgcttcttcc caaagatgaa cgcggcgtta tgtcactaac gacgtgcacc 60aacttgcggg aattctcatg ggatatttgc tcaagacgac ggtaaaatga gcaaatatgg 120cacgatcctc aattgtcatg ggatatttgc tcaagacgac ggtaaaatga gcaaatatgg 180cacgatcctc aatgtcatgg gatatttgct caagacgacg gtaaaatgag caaatatggc 240acgatcctga attccaccgc acgccttttt tctgaagccc actttcgtgg actttgccat 300atatgcaaaa ttcatgaagt gtgataccaa gtcagcatac acctcactag ggtagtttct 360ttggttgtat tgatcatttg gttcatcgtg gttcattaat tttttttctc cattgctttc 420tggctttgat cttactatca tttggatttt tgtcgaaggt tgtagaattg tatgtgacaa 480gtggcaccaa gcatatataa aaaaaaaaag cattatcttc ctaccagagt tgattgttaa 540aaacgtattt atagcaaacg caattgtaat taattcttat tttgtatctt ttcttccctt 600gtctcaatct tttattttta ttttattttt cttttcttag tttctttcat aacaccaagc 660aactaatact ataacataca ataata 68678747DNAArtificial SequenceSynthetic pCUR17Synthetic pCUR17 78gctcagcatc tgcttcttcc caaagatgaa cgcggcgtta tgtcactaac gacgtgcacc 60aacttgcggg aattctcatg ggatatttgc tcaagacgac ggtaaaatga gcaaatatgg 120cacgatcctc aattgtcatg ggatatttgc tcaagacgac ggtaaaatga gcaaatatgg 180cacgatcctc aattctcatg ggatatttgc tcaagacgac ggtaaaatga gcaaatatgg 240cacgatcctc aattctcatg ggatatttgc tcaagacgac ggtaaaatga gcaaatatgg 300cacgatcctg aattccaccg cacgcctttt ttctgaagcc cactttcgtg gactttgcca 360tatatgcaaa attcatgaag tgtgatacca agtcagcata cacctcacta gggtagtttc 420tttggttgta ttgatcattt ggttcatcgt ggttcattaa ttttttttct ccattgcttt 480ctggctttga tcttactatc atttggattt ttgtcgaagg ttgtagaatt gtatgtgaca 540agtggcacca agcatatata aaaaaaaaaa gcattatctt cctaccagag ttgattgtta 600aaaacgtatt tatagcaaac gcaattgtaa ttaattctta ttttgtatct tttcttccct 660tgtctcaatc ttttattttt attttatttt tcttttctta gtttctttca taacaccaag 720caactaatac tataacatac aataata 74779500DNASaccharomyces cerevisiaepLYS1pLYS1 79gcaagttaac attagggaga acgtggggcc ttcctccatg agtgcagagc aattgaagat 60gtttagaggt ttaaaggaga ataaccagtt gctggatagc tctgtgccag ctacagttta 120tgccaaattg gcccttcatg gtattcctga cggtgttaat ggacagtact tgagctataa 180tgaccctgcc ttggcggact ttatgccttg aggatagcag gtacatataa attgttacat 240actaagtcga tgagtcaaaa aagactctta tacatttata cattttgcat tattattttt 300tttttccagc ggaatttgga attccgctct caaccgccaa aattcccctg cgatttcagc 360gacaaagagt cataaagtca tcctcgagaa accacgatga aatatataaa aagcccatct 420tccctgacgg aaactggtat tttaggaggc ataccataag ataacaacga aaacgcttta 480tttttcacac aaccgcaaaa 50080650DNASaccharomyces cerevisiaepLYS4pLYS4 80ttgaaaaatg cgaagttgaa gtgccataga agagaaacag cccacacagg ggagaagccc 60actggaaagg gggcactgac caactttaaa taggaaacag aagataccac aagccagcga 120tacaacagca ccaaacaccg aaaagaatag ccaaagctgt cctctggtgt tggaaaaact 180ggaaaaaacg caactgcgtt ggctgctacg gtgaaaaatt ttcctatgac ttttttcact 240gcttgttcgt gcgaaattac cgcaaacccg gtaaaatgta cacgtatcaa gtgataaaca 300atttcgtgtc aagtgagcag aatggagcga tttggaaaaa aaaaattttt attgtttttt 360cccccgggat tttgctcgag atgactgaaa ttttgtaatc gatgagtcta taccagaggc 420agcaaatatc accaacatac acaggtatac acaatctcat gtccacacac acgtacagac 480acgcacatat atatatatat atatatatcc ccataggtat ttatatatac aaaagaatcc 540tcgtgtgttt gtgtgtgcaa tagctagttt tgcgctgcct cttatagtag acaatatcac 600tttttcaata aaatagaact tgcaaggaaa caaaattgta tcgcttcaag 65081500DNASaccharomyces cerevisiaepLYS9pLYS9 81acatatgcaa gagtcttatg tatcgtatct aagtgccacg taggggattc ccatcatttg 60atgatttcca aatataatac ctgtagagag cggtggagca aaagtcaaat tttaatcgca 120actgcagaca agtcaagctg aggaaattgt ggatgatctc ttgtttcttt tgatattcac 180cacaacagaa gtgaagagtg tgattgcggt tactactgac cacgaagcaa tgcgtttagt 240agtgaaaaga attactcata ctctggaatc gaaattccgt tggaaaaatt cgctttgtag 300tgaaaaataa agatgtcaat aaagggtatt gagaatttcc aatggaatta tcagcaatag 360atgatagaaa gtagcacaga atttggctta atggtatata aaccgtaggg tcctggtaaa 420attacatggg aaggatcctt aggcagtagg gaaaacttat caggacaatt gagttatatt 480aacgtattat atattttaat 50082494DNAArtificial SequenceSynthetic pLYR1pSynthetic LYR1p 82ttatattgaa ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag aagtttaata 60atcatattac atggcattac caccatatac atatccatat ctaatcttac ttatatgttg 120tggaaatgta aagagcccga attcctcata ctctggaatc gaaattccgt tggaaaaatt 180cgctttgtag tgaaaaataa agatgtcaat aaagggtatt gagaatttcc aatggaatta 240tcagcaatag atgatagaaa gaattcggat gataatgcga ttagtttttt agccttattt 300ctggggtaat taatcagcga agcgatgatt tttgatctat taacagatat ataaatggaa 360aagctgcata accactttaa ctaatacttt caacattttc agtttgtatt acttcttatt 420caaatgtcat aaaagtatca acaaaaaatt gttaatatac ctctatactt taacgtcaag 480gagaaaaaac tata 49483494DNAArtificial SequenceSynthetic pLYR2pSynthetic pLYR2p 83ttatattgaa ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag aagtttaata 60atcatattac atggcattac caccatatac atatccatat ctaatcttac ttatatgttg 120tggaaatgta aagagcccga attctttcta tcatctattg ctgataattc cattggaaat 180tctcaatacc ctttattgac atctttattt ttcactacaa agcgaatttt tccaacggaa 240tttcgattcc agagtatgag gaattcggat gataatgcga ttagtttttt agccttattt 300ctggggtaat taatcagcga agcgatgatt tttgatctat taacagatat ataaatggaa 360aagctgcata accactttaa ctaatacttt caacattttc agtttgtatt acttcttatt 420caaatgtcat aaaagtatca acaaaaaatt gttaatatac ctctatactt taacgtcaag 480gagaaaaaac tata 49484616DNAArtificial SequenceSynthetic pLYR3pSynthetic pLYR3p 84ttatattgaa ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag aagtttaata 60atcatattac atggcattac caccatatac atatccatat ctaatcttac ttatatgttg 120tggaaatgta aagagcccga attcctcata ctctggaatc gaaattccgt tggaaaaatt 180cgctttgtag tgaaaaataa agatgtcaat aaagggtatt gagaatttcc aatggaatta 240tcagcaatag atgatagaaa gaattcctca tactctggaa tcgaaattcc gttggaaaaa 300ttcgctttgt agtgaaaaat aaagatgtca ataaagggta ttgagaattt ccaatggaat 360tatcagcaat agatgataga aagaattcgg atgataatgc gattagtttt ttagccttat 420ttctggggta attaatcagc gaagcgatga tttttgatct attaacagat atataaatgg 480aaaagctgca taaccacttt aactaatact ttcaacattt tcagtttgta ttacttctta 540ttcaaatgtc ataaaagtat caacaaaaaa ttgttaatat acctctatac tttaacgtca 600aggagaaaaa actata 61685616DNAArtificial SequenceSynthetic pLYR4pSynthetic LYR4p 85ttatattgaa ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag aagtttaata 60atcatattac atggcattac caccatatac atatccatat ctaatcttac ttatatgttg 120tggaaatgta aagagcccga attctttcta tcatctattg ctgataattc cattggaaat 180tctcaatacc ctttattgac atctttattt ttcactacaa agcgaatttt tccaacggaa 240tttcgattcc agagtatgag gaattctttc tatcatctat tgctgataat tccattggaa 300attctcaata ccctttattg acatctttat ttttcactac aaagcgaatt tttccaacgg 360aatttcgatt ccagagtatg aggaattcgg atgataatgc gattagtttt ttagccttat 420ttctggggta attaatcagc gaagcgatga tttttgatct attaacagat atataaatgg 480aaaagctgca taaccacttt aactaatact ttcaacattt tcagtttgta ttacttctta 540ttcaaatgtc ataaaagtat caacaaaaaa ttgttaatat

acctctatac tttaacgtca 600aggagaaaaa actata 61686738DNAArtificial SequenceSynthetic pLYR5pSynthetic pLYR5p 86ttatattgaa ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag aagtttaata 60atcatattac atggcattac caccatatac atatccatat ctaatcttac ttatatgttg 120tggaaatgta aagagcccga attctttcta tcatctattg ctgataattc cattggaaat 180tctcaatacc ctttattgac atctttattt ttcactacaa agcgaatttt tccaacggaa 240tttcgattcc agagtatgag gaattctttc tatcatctat tgctgataat tccattggaa 300attctcaata ccctttattg acatctttat ttttcactac aaagcgaatt tttccaacgg 360aatttcgatt ccagagtatg aggaattctt tctatcatct attgctgata attccattgg 420aaattctcaa taccctttat tgacatcttt atttttcact acaaagcgaa tttttccaac 480ggaatttcga ttccagagta tgaggaattc ggatgataat gcgattagtt ttttagcctt 540atttctgggg taattaatca gcgaagcgat gatttttgat ctattaacag atatataaat 600ggaaaagctg cataaccact ttaactaata ctttcaacat tttcagtttg tattacttct 660tattcaaatg tcataaaagt atcaacaaaa aattgttaat atacctctat actttaacgt 720caaggagaaa aaactata 738871104DNAArtificial SequenceSynthetic pLYR6pSynthetic pLYR6p 87ttatattgaa ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag aagtttaata 60atcatattac atggcattac caccatatac atatccatat ctaatcttac ttatatgttg 120tggaaatgta aagagcccga attgctcata ctctggaatc gaaattccgt tggaaaaatt 180cgctttgtag tgaaaaataa agatgtcaat aaagggtatt gagaatttcc aatggaatta 240tcagcaatag atgatagaaa gaattcctca tactctggaa tcgaaattcc gttggaaaaa 300ttcgctttgt agtgaaaaat aaagatgtca ataaagggta ttgagaattt ccaatggaat 360tatcagcaat agatgataga aagaattcct catactctgg aatcgaaatt ccgttggaaa 420aattcgcttt gtagtgaaaa ataaagatgt caataaaggg tattgagaat ttccaatgga 480attatcagca atagatgata gaaacaattg ctcatactct ggaatcgaaa ttccgttgga 540aaaattcgct ttgtagtgaa aaataaagat gtcaataaag ggtattgaga atttccaatg 600gaattatcag caatagatga tagaaagaat tcctcatact ctggaatcga aattccgttg 660gaaaaattcg ctttgtagtg aaaaataaag atgtcaataa agggtattga gaatttccaa 720tggaattatc agcaatagat gatagaaaga attcctcata ctctggaatc gaaattccgt 780tggaaaaatt cgctttgtag tgaaaaataa agatgtcaat aaagggtatt gagaatttcc 840aatggaatta tcagcaatag atgatagaaa caattcggat gataatgcga ttagtttttt 900agccttattt ctggggtaat taatcagcga agcgatgatt tttgatctat taacagatat 960ataaatggaa aagctgcata accactttaa ctaatacttt caacattttc agtttgtatt 1020acttcttatt caaatgtcat aaaagtatca acaaaaaatt gttaatatac ctctatactt 1080taacgtcaag gagaaaaaac tata 1104881836DNAArtificial SequenceSynthetic pLYR7pSynthetic pLYR7p 88ttatattgaa ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag aagtttaata 60atcatattac atggcattac caccatatac atatccatat ctaatcttac ttatatgttg 120tggaaatgta aagagcccga attgtttcta tcatctattg ctgataattc cattggaaat 180tctcaatacc ctttattgac atctttattt ttcactacaa agcgaatttt tccaacggaa 240tttcgattcc agagtatgag gaattctttc tatcatctat tgctgataat tccattggaa 300attctcaata ccctttattg acatctttat ttttcactac aaagcgaatt tttccaacgg 360aatttcgatt ccagagtatg aggaattctt tctatcatct attgctgata attccattgg 420aaattctcaa taccctttat tgacatcttt atttttcact acaaagcgaa tttttccaac 480ggaatttcga ttccagagta tgagcaattg tttctatcat ctattgctga taattccatt 540ggaaattctc aatacccttt attgacatct ttatttttca ctacaaagcg aatttttcca 600acggaatttc gattccagag tatgaggaat tctttctatc atctattgct gataattcca 660ttggaaattc tcaataccct ttattgacat ctttattttt cactacaaag cgaatttttc 720caacggaatt tcgattccag agtatgagga attctttcta tcatctattg ctgataattc 780cattggaaat tctcaatacc ctttattgac atctttattt ttcactacaa agcgaatttt 840tccaacggaa tttcgattcc agagtatgag caattgtttc tatcatctat tgctgataat 900tccattggaa attctcaata ccctttattg acatctttat ttttcactac aaagcgaatt 960tttccaacgg aatttcgatt ccagagtatg aggaattctt tctatcatct attgctgata 1020attccattgg aaattctcaa taccctttat tgacatcttt atttttcact acaaagcgaa 1080tttttccaac ggaatttcga ttccagagta tgaggaattc tttctatcat ctattgctga 1140taattccatt ggaaattctc aatacccttt attgacatct ttatttttca ctacaaagcg 1200aatttttcca acggaatttc gattccagag tatgagcaat tgtttctatc atctattgct 1260gataattcca ttggaaattc tcaataccct ttattgacat ctttattttt cactacaaag 1320cgaatttttc caacggaatt tcgattccag agtatgagga attctttcta tcatctattg 1380ctgataattc cattggaaat tctcaatacc ctttattgac atctttattt ttcactacaa 1440agcgaatttt tccaacggaa tttcgattcc agagtatgag gaattctttc tatcatctat 1500tgctgataat tccattggaa attctcaata ccctttattg acatctttat ttttcactac 1560aaagcgaatt tttccaacgg aatttcgatt ccagagtatg agcaattcgg atgataatgc 1620gattagtttt ttagccttat ttctggggta attaatcagc gaagcgatga tttttgatct 1680attaacagat atataaatgg aaaagctgca taaccacttt aactaatact ttcaacattt 1740tcagtttgta ttacttctta ttcaaatgtc ataaaagtat caacaaaaaa ttgttaatat 1800acctctatac tttaacgtca aggagaaaaa actata 183689981DNAArtificial SequenceSynthetic pLYR8Synthetic pLYR8 89ttacattatc aatccttgcg tttcagcttc cactaattta gatgactatt tctcatcatt 60tgcgtcatct tctaacaccg tatatgataa tatactagta acgtaaatac tagttagtag 120atgatagttg atttttattc caacactaag aaataatttc gccatttctt gaatgtattt 180aaagatattt aatgctataa tagacattta aatccaattc ttccaacata caatgggagt 240ttggccgagt ggtttaaggc gtcagattta ggtggattta acctctaaaa tctctgatat 300cttcggatgc aagggttcga atcccttagc tctcattatt ttttgctttt tctcttgaat 360tgctcatact ctggaatcga aattccgttg gaaaaattcg ctttgtagtg aaaaataaag 420atgtcaataa agggtattga gaatttccaa tggaattatc agcaatagat gatagaaaga 480attcctcata ctctggaatc gaaattccgt tggaaaaatt cgctttgtag tgaaaaataa 540agatgtcaat aaagggtatt gagaatttcc aatggaatta tcagcaatag atgatagaaa 600gaattcctca tactctggaa tcgaaattcc gttggaaaaa ttcgctttgt agtgaaaaat 660aaagatgtca ataaagggta ttgagaattt ccaatggaat tatcagcaat agatgataga 720aacaattcta attaagttag tcaaggcgcc atcctcatga aaactgtgta acataataac 780cgaagtgtcg aaaaggtggc accttgtcca attgaacacg ctcgatgaaa aaaataagat 840atatataagg ttaagtaaag cgtctgttag aaaggaagtt tttccttttt cttgctctct 900tgtcttttca tctactattt ccttcgtgta atacagggtc gtcagataca tagatacaat 960tctattaccc ccatccatac a 98190981DNAArtificial SequenceSynthetic pLYR9Synthetic pLYR9 90ttacattatc aatccttgcg tttcagcttc cactaattta gatgactatt tctcatcatt 60tgcgtcatct tctaacaccg tatatgataa tatactagta acgtaaatac tagttagtag 120atgatagttg atttttattc caacactaag aaataatttc gccatttctt gaatgtattt 180aaagatattt aatgctataa tagacattta aatccaattc ttccaacata caatgggagt 240ttggccgagt ggtttaaggc gtcagattta ggtggattta acctctaaaa tctctgatat 300cttcggatgc aagggttcga atcccttagc tctcattatt ttttgctttt tctcttgaat 360tgtttctatc atctattgct gataattcca ttggaaattc tcaataccct ttattgacat 420ctttattttt cactacaaag cgaatttttc caacggaatt tcgattccag agtatgagga 480attctttcta tcatctattg ctgataattc cattggaaat tctcaatacc ctttattgac 540atctttattt ttcactacaa agcgaatttt tccaacggaa tttcgattcc agagtatgag 600gaattctttc tatcatctat tgctgataat tccattggaa attctcaata ccctttattg 660acatctttat ttttcactac aaagcgaatt tttccaacgg aatttcgatt ccagagtatg 720agcaattcta attaagttag tcaaggcgcc atcctcatga aaactgtgta acataataac 780cgaagtgtcg aaaaggtggc accttgtcca attgaacacg ctcgatgaaa aaaataagat 840atatataagg ttaagtaaag cgtctgttag aaaggaagtt tttccttttt cttgctctct 900tgtcttttca tctactattt ccttcgtgta atacagggtc gtcagataca tagatacaat 960tctattaccc ccatccatac a 981911225DNAArtificial SequenceSynthetic pLYR10Synthetic pLYR10 91ttacattatc aatccttgcg tttcagcttc cactaattta gatgactatt tctcatcatt 60tgcgtcatct tctaacaccg tatatgataa tatactagta acgtaaatac tagttagtag 120atgatagttg atttttattc caacactaag aaataatttc gccatttctt gaatgtattt 180aaagatattt aatgctataa tagacattta aatccaattc ttccaacata caatgggagt 240ttggccgagt ggtttaaggc gtcagattta ggtggattta acctctaaaa tctctgatat 300cttcggatgc aagggttcga atcccttagc tctcattatt ttttgctttt tctcttgaat 360tgtttctatc atctattgct gataattcca ttggaaattc tcaataccct ttattgacat 420ctttattttt cactacaaag cgaatttttc caacggaatt tcgattccag agtatgagga 480attctttcta tcatctattg ctgataattc cattggaaat tctcaatacc ctttattgac 540atctttattt ttcactacaa agcgaatttt tccaacggaa tttcgattcc agagtatgag 600gaattctttc tatcatctat tgctgataat tccattggaa attctcaata ccctttattg 660acatctttat ttttcactac aaagcgaatt tttccaacgg aatttcgatt ccagagtatg 720aggaattctt tctatcatct attgctgata attccattgg aaattctcaa taccctttat 780tgacatcttt atttttcact acaaagcgaa tttttccaac ggaatttcga ttccagagta 840tgaggaattc tttctatcat ctattgctga taattccatt ggaaattctc aatacccttt 900attgacatct ttatttttca ctacaaagcg aatttttcca acggaatttc gattccagag 960tatgagcaat tctaattaag ttagtcaagg cgccatcctc atgaaaactg tgtaacataa 1020taaccgaagt gtcgaaaagg tggcaccttg tccaattgaa cacgctcgat gaaaaaaata 1080agatatatat aaggttaagt aaagcgtctg ttagaaagga agtttttcct ttttcttgct 1140ctcttgtctt ttcatctact atttccttcg tgtaatacag ggtcgtcaga tacatagata 1200caattctatt acccccatcc ataca 1225921347DNAArtificial SequenceSynthetic pLYR11Synthetic pLYR11 92ttacattatc aatccttgcg tttcagcttc cactaattta gatgactatt tctcatcatt 60tgcgtcatct tctaacaccg tatatgataa tatactagta acgtaaatac tagttagtag 120atgatagttg atttttattc caacactaag aaataatttc gccatttctt gaatgtattt 180aaagatattt aatgctataa tagacattta aatccaattc ttccaacata caatgggagt 240ttggccgagt ggtttaaggc gtcagattta ggtggattta acctctaaaa tctctgatat 300cttcggatgc aagggttcga atcccttagc tctcattatt ttttgctttt tctcttgaat 360tgctcatact ctggaatcga aattccgttg gaaaaattcg ctttgtagtg aaaaataaag 420atgtcaataa agggtattga gaatttccaa tggaattatc agcaatagat gatagaaaga 480attcctcata ctctggaatc gaaattccgt tggaaaaatt cgctttgtag tgaaaaataa 540agatgtcaat aaagggtatt gagaatttcc aatggaatta tcagcaatag atgatagaaa 600gaattcctca tactctggaa tcgaaattcc gttggaaaaa ttcgctttgt agtgaaaaat 660aaagatgtca ataaagggta ttgagaattt ccaatggaat tatcagcaat agatgataga 720aagaattcct catactctgg aatcgaaatt ccgttggaaa aattcgcttt gtagtgaaaa 780ataaagatgt caataaaggg tattgagaat ttccaatgga attatcagca atagatgata 840gaaagaattc ctcatactct ggaatcgaaa ttccgttgga aaaattcgct ttgtagtgaa 900aaataaagat gtcaataaag ggtattgaga atttccaatg gaattatcag caatagatga 960tagaaagaat tcctcatact ctggaatcga aattccgttg gaaaaattcg ctttgtagtg 1020aaaaataaag atgtcaataa agggtattga gaatttccaa tggaattatc agcaatagat 1080gatagaaaca attctaatta agttagtcaa ggcgccatcc tcatgaaaac tgtgtaacat 1140aataaccgaa gtgtcgaaaa ggtggcacct tgtccaattg aacacgctcg atgaaaaaaa 1200taagatatat ataaggttaa gtaaagcgtc tgttagaaag gaagtttttc ctttttcttg 1260ctctcttgtc ttttcatcta ctatttcctt cgtgtaatac agggtcgtca gatacataga 1320tacaattcta ttacccccat ccataca 134793686DNASaccharomyces cerevisiaepMET17pMET17 93ttacattatc aatccttgcg tttcagcttc cactaattta gatgactatt tctcatcatt 60tgcgtcatct tctaacaccg tatatgataa tatactagta acgtaaatac tagttagtag 120atgatagttg atttttattc caacactaag aaataatttc gccatttctt gaatgtattt 180aaagatattt aatgctataa tagacattta aatccaattc ttccaacata caatgggagt 240ttggccgagt ggtttaaggc gtcagattta ggtggattta acctctaaaa tctctgatat 300cttcggatgc aagggttcga atcccttagc tctcattatt ttttgctttt tctcttgagg 360tcacatgatc gcaaaatggc aaatggcacg tgaagctgtc gatattgggg aactgtggtg 420gttggcaaat gactaattaa gttagtcaag gcgccatcct catgaaaact gtgtaacata 480ataaccgaag tgtcgaaaag gtggcacctt gtccaattga acacgctcga tgaaaaaaat 540aagatatata taaggttaag taaagcgtct gttagaaagg aagtttttcc tttttcttgc 600tctcttgtct tttcatctac tatttccttc gtgtaataca gggtcgtcag atacatagat 660acaattctat tacccccatc cataca 68694510DNASaccharomyces cerevisiaepMET6pMET6 94ccacaggaaa tatttcacgt gacttacaaa cagagtcgta cgtcaggacc ggagtcaggt 60gaaaaaatgt gggccggtaa agggaaaaaa ccagaaacgg gactactatc gaactcgttt 120agtcgcgaac gtgcaaaagg ccaatatttt tcgctagagt catcgcagtc atggcagctc 180tttcgctcta tctcccggtc gcaaaactgt ggtagtcata gctcgttctg ctcaattgag 240aactgtgaat gtgaatatgg aacaaatgcg atagatgcac taatttaagg gaagctagct 300agttttccca actgcgaaag aaaaaaagga aagaaaaaaa aattctatat aagtgataga 360tatttccatc tttactagca ttagtttctc ttttacgtat tcaatatttt tgttaaactc 420ttcctttatc ataaaaaagc aagcatctaa gagcattgac aacactctaa gaaacaaaat 480accaatataa tttcaaagta catatcaaaa 51095508DNASaccharomyces cerevisiaepMET14pMET14 95cctatgcatg tttagagcaa gcgcctttgt gagccctccc ggttacgacg ccttggcaat 60gtagcagata actctgcact tctagaatca ttccactacg acatttggct catcaccagc 120tcgcgagaaa tgtaaataag ccaacaacca agaatgcgta acattaaaga atacagttgc 180tttcatttcg gcgtgatggt acggcaccca cggttcctta cattattctc gaaaaatagc 240tgcacgcttt tccaggaata aaagaccgtg ccactaattt cacgtgatca atatatttac 300aagccacctc aaaaaatgtg gcaatggaga agaggatgaa cgactcaata tgacttcaac 360ttcatgaatt tgtcaaaata tctatataag atgcaaaatt tctatacaac atcagttgcg 420tatccgttaa tgtcgttcat tttctctctt tgttcgaact tgacatcaag aaaagttgga 480attatttctc caagcacact gtacacca 50896552DNASaccharomyces cerevisiaepMET3pMET3 96aacgatatgt acgtagtggt ataaggtgag ggggtccaca gatataacat cgtttaattt 60agtactaaca gagacttttg tcacaactac atataagtgt acaaatatag tacagatatg 120acacacttgt agcgccaacg cgcatcctac ggattgctga cagaaaaaaa ggtcacgtga 180ccagaaaagt cacgtgtaat tttgtaactc accgcattct agcggtccct gtcgtgcaca 240ctgcactcaa caccataaac cttagcaacc tccaaaggaa atcaccgtat aacaaagcca 300cagttttaca acttagtctc ttatgaagtt acttaccaat gagaaataga ggctctttct 360cgacaaatat gaatatggat atatatatat atatatatat atatatatat atatatatgt 420aaacttggtt cttttttagc ttgtgatctc tagcttgggt ctctctctgt cgtaacagtt 480gtgatatcgt ttcttaacaa ttgaaaagga actaagaaag tataataata acaagaataa 540agtataatta ac 55297363DNASaccharomyces cerevisiaepSAM1pSAM1 97gaaacggacg taagacggaa atagaatttg aagataaagt tatatatcac tacacacgaa 60tactttcttt tttttttttc acaggaaaac tgtggtggcg cccttgccta ctagtgcatt 120tcttttttcg ggttcttgtc tcgacgaaat tttagcctca tcgtagtttt tcactctggt 180atcgatgaaa aagggaagag taaaaagttt tccgtttagt acttaatggg attggtttgg 240gacgtatata tcgactggtg ttgtctgtta ttcatcgttg tttttcggtt agcttcgaaa 300aaaaaataga gtaaaaacca ggaatttacc ctaaaaacaa gaaaaaataa gataaacgaa 360aat 36398500DNASaccharomyces cerevisiaepSAM2pSAM2 98gagctttgct ctattatata agataaaata tgcactaaaa gtttgcattt ctttacataa 60ctaaaactaa gacattatgc atagcttacc tgatcaaaaa gtatgtaaac ttgttaacat 120cttcacatgt gattcatctg gtcgtacttt cttgcggtgc agtgtaatat ttctacccac 180gtgactataa ttgagcttga aaactgtggc gtttttccac cgatgggtcc acgccagata 240ttaaccgaag ccaaaatacc gatgaaattt ctgagatagc tcttgtaaac gacgtcaaat 300cttcatatgc aaggagatct tgatttcttt ttggtagtca tctgtcgtct tgaggcgtat 360aagaaggagg ttatatctgt cctttctaca aagtattttc gagaatcttg cttctgcccc 420ttttttcttt ttttaaaagg tttaaaaaac ataactgtct tcaatatatc cagtatttac 480gacaatatac aaacataatc 50099300DNASaccharomyces cerevisiaetTDH2tTHD2 99atttaactcc ttaagttact ttaatgattt agtttttatt attaataatt catgctcatg 60acatctcata tacacgttta taaaacttaa atagattgaa aatgtattaa agattcctca 120gggattcgat ttttttggaa gtttttgttt ttttttcctt gagatgctgt agtatttggg 180aacaattata caatcgaaag atatatgctt acattcgacc gttttagccg tgatcattat 240cctatagtaa cataacctga agcataactg acactactat catcaatact tgtcacatga 300100300DNASaccharomyces cerevisiaetCYC1tCYC1 100acaggcccct tttcctttgt cgatatcatg taattagtta tgtcacgctt acattcacgc 60cctcctccca catccgctct aaccgaaaag gaaggagtta gacaacctga agtctaggtc 120cctatttatt ttttttaata gttatgttag tattaagaac gttatttata tttcaaattt 180ttcttttttt tctgtacaaa cgcgtgtacg catgtaacat tatactgaaa accttgcttg 240agaaggtttt gggacgctcg aaggctttaa tttgcaagct tcgcagttta cactctcatc 300101300DNASaccharomyces cerevisiaetTDH3tTDH3 101gtgaatttac tttaaatctt gcatttaaat aaattttctt tttatagctt tatgacttag 60tttcaattta tatactattt taatgacatt ttcgattcat tgattgaaag ctttgtgttt 120tttcttgatg cgctattgca ttgttcttgt ctttttcgcc acatgtaata tctgtagtag 180atacctgata cattgtggat gctgagtgaa attttagtta ataatggagg cgctcttaat 240aattttgggg atattggctt ttttttttaa agtttacaaa tgaatttttt ccgccaggat 300102354DNASaccharomyces cerevisiaetADH1tADH1 102actagttcta gagcggccgc caccgcggtg ggcgaatttc ttatgattta tgatttttat 60tattaaataa gttataaaaa aaataagtgt atacaaattt taaagtgact cttaggtttt 120aaaacgaaaa ttcttattct tgagtaactc tttcctgtag gtcaggttgc tttctcaggt 180atagcatgag gtcgctctta ttgaccacac ctctaccggc atgccgagca aatgcctgca 240aatcgctccc catttcaccc aattgtagat atgctaactc cagcaatgag ttgatgaatc 300tcggtgtgta ttttatgtcc tcagaggaca acacctgttg taatcgttct tcca 354103301DNASaccharomyces cerevisiaetADH2tADH2 103gcggatctct tatgtcttta cgatttatag ttttcattat caagtatgcc tatattagta 60tatagcatct ttagatgaca gtgttcgaag tttcacgaat aaaagataat attctacttt 120ttgctcccac cgcgtttgct agcacgagtg aacaccatcc ctcgcctgtg agttgtaccc 180attcctctaa actgtagaca tggtagcttc agcagtgttc gttatgtacg gcatcctcca 240acaaacagtc ggttatagtt tgtcctgctc ctctgaatcg tctccctcga tatttctcat 300t 301104299DNASaccharomyces cerevisiaetTPI1tTPI1 104gattaatata attatataaa aatattatct tcttttcttt atatctagtg ttatgtaaaa 60taaattgatg actacggaaa gcttttttat attgtttctt tttcattctg agccacttaa 120atttcgtgaa tgttcttgta agggacggta gatttacaag tgatacaaca aaaagcaagg 180cgctttttct aataaaaaga agaaaagcat ttaacaattg aacacctcta tatcaacgaa 240gaatattact ttgtctctaa atccttgtaa aatgtgtacg atctctatat gggttactc 299105299DNASaccharomyces cerevisiaetMET17tMET17 105gtgtgcgtaa tgagttgtaa aattatgtat aaacctactt tctctcacaa gtactatact 60tttataaaac gaactttatt gaaatgaata tccttttttt cccttgttac atgtcgtgac 120tcgtactttg aacctaaatt gttctaacat caaagaacag tgttaattcg cagtcgagaa 180gaaaaatatg gtgaacaaga ctcatctact tcatgagact actttacgcc tcctataaag 240ctgtcacact ggataaattt attgtaggac caagttacaa aagaggatga tggaggttt

299106305DNASaccharomyces cerevisiaetENO2tENO2 106ggatcctaaa gtgcttttaa ctaagaatta ttagtctttt ctgcttattt tttcatcata 60gtttagaaca ctttatatta acgaatagtt tatgaatcta tttaggttta aaaattgata 120cagttttata agttactttt tcaaagactc gtgctgtcta ttgcataatg cactggaagg 180ggaaaaaaaa ggtgcacacg cgtggctttt tcttgaattt gcagtttgaa aaataactac 240atggatgata agaaaacatg gagtacagtc actttgagaa ccttcaatca gctggtaacg 300tcttc 305107300DNASaccharomyces cerevisiaetMET3tMET3 107tcgtcataaa atgctcccat ctcaaaagta gggcaaaatt catgatcgac cgcgcaaaat 60aaatagattt gcaaataagt tttgtatgta catttattaa tatatataat atatcaaaag 120aaaaaaatca aaaaaaaaaa aaaaaaaaaa ttgcactctt attcagtcat caattacaaa 180acctagagat agcgatggtg catattcaat aaaaaactcc ttatactgtc gagaaagctt 240attattggta cttctcgaag atactaaaaa aggttaattt ttggagacgg aggcaatagc 300108301DNASaccharomyces cerevisiaetPGK1tPGK1 108attgaattga attgaaatcg atagatcaat ttttttcttt tctctttccc catcctttac 60gctaaaataa tagtttattt tattttttga atatttttta tttatatacg tatatataga 120ctattattta tcttttaatg attattaaga tttttattaa aaaaaaattc gctcctcttt 180taatgccttt atgcagtttt tttttcccat tcgatatttc tatgttcggg ttcagcgtat 240tttaagttta ataactcgaa aattctgcgt tcgttaaagc tttcgagaag gatattattt 300a 301109300DNASaccharomyces cerevisiaetDIT1tDIT1 109taaagtaaga gcgctacatt ggtctacctt tttgttcttt tacttaaaca ttagttagtt 60cgttttcttt ttctcatttt tttatgtttc ccccccaaag ttctgatttt ataatatttt 120atttcacaca attccattta acagaggggg aatagattct ttagcttaga aaattagtga 180tcaatatata tttgcctttc ttttcatctt ttcagtgata ttaatggttt cgagacactg 240caatggccct agttgtctaa gaggatagat gttactgtca aagatgatat tttgaatttc 300110300DNASaccharomyces cerevisiaetRPL3tRPL3 110gaagttttgt tagaaaataa atcatttttt aattgagcat tcttattcct attttattta 60aatagtttta tgtattgtta gctacataca acagtttaaa tcaaattttc tttttcccaa 120gtccaaaatg gaggtttatt ttgatgaccc gcatgcgatt atgttttgaa agtataagac 180tacatacatg tacatatatt taaacatgta aacccgtcca ttatattgct tactttcttc 240ttttttgccg ttttgacttg gacctctggt ttgctatttc cttacaatct ttgctacaat 300111300DNASaccharomyces cerevisiaetRPL41BtRPL41B 111gcggattgag agcaaatcgt taagttcagg tcaagtaaaa attgatttcg aaaactaatt 60tctcttatac aatcctttga ttggaccgtc atcctttcga atataagatt ttgttaagaa 120tattttagac agagatctac tttatattta atatctagat attacataat ttcctctcta 180ataaaatatc attaataaaa taaaaatgaa gcgatttgat tttgtgttgt caacttagtt 240tgccgctatg cctcttgggt aatgctatta ttgaatcgaa gggctttatt atattaccct 300112300DNASaccharomyces cerevisiaetRPL15AtRPL15A 112gctggttgat ggaaaatata attttattgg gcaaactttt gtttatctga tgtgttttat 60actattatct ttttaattaa tgattctata tacaaacctg tatatttttt ctttaaccaa 120tttttttttt tatagaccta gagctgtact tttattctgc tatcaagcaa acccctaccc 180cctcttctca atcctcccct caggcagaac ttatctacct gtatcaagga gcggacgagg 240gagtcctaat tgttctacgt ataccaatgc tagcagctta cataggtggt ggcactacca 300113300DNASaccharomyces cerevisiaetIDP1tIDP1 113tcgaatttac gtagcccaat ctaccacttt tttttttcat tttttaaagt gttatactta 60gttatgctct aggataatga actacttttt tttttttttt tttactgtta tcataaatat 120atatacctta ttgttgtttg caaccgtcgg ttaattcctt atcaaggttc cccaagttcg 180gatcattacc atcaatttcc aacattttca tgagttcttc ttcttcatta ccgtgtttta 240gggggctgtt cgcacttcta atagggctat caccaagctg ttctaattcg tccaaaagtt 3001141089DNAKluveromyces lactisLeu2Leu2 114atgtctaaga atatcgttgt cctaccgggt gatcacgtcg gtaaagaagt tactgacgaa 60gctattaagg tcttgaatgc cattgctgaa gtccgtccag aaattaagtt caatttccaa 120catcacttga tcgggggtgc tgccatcgat gccactggca ctcctttacc agatgaagct 180ctagaagcct ctaagaaagc cgatgctgtc ttactaggtg ctgttggtgg tccaaaatgg 240ggtacgggcg cagttagacc agaacaaggt ctattgaaga tcagaaagga attgggtcta 300tacgccaact tgagaccatg taactttgct tctgattctt tactagatct ttctcctttg 360aagcctgaat atgcaaaggg taccgatttc gtcgtcgtta gagaattggt tggtggtatc 420tactttggtg aaagaaaaga agatgaaggt gacggagttg cttgggactc tgagaaatac 480agtgttcctg aagttcaaag aattacaaga atggctgctt tcttggcatt gcaacaaaac 540ccaccattac caatctggtc tcttgacaag gctaacgtgc ttgcctcttc cagattgtgg 600agaaagactg ttgaagaaac catcaagact gagttcccac aattaactgt tcagcaccaa 660ttgatcgact ctgctgctat gattttggtt aaatcaccaa ctaagctaaa cggtgttgtt 720attaccaaca acatgtttgg tgatattatc tccgatgaag cctctgttat tccaggttct 780ttgggtttat taccttctgc atctctagct tccctacctg acactaacaa ggcattcggt 840ttgtacgaac catgtcatgg ttctgcccca gatttaccag caaacaaggt taacccaatt 900gctaccatct tatctgcagc tatgatgttg aagttatcct tggatttggt tgaagaaggt 960agggctcttg aagaagctgt tagaaatgtc ttggatgcag gtgtcagaac cggtgacctt 1020ggtggttcta actctaccac tgaggttggc gatgctatcg ccaaggctgt caaggaaatc 1080ttggcttaa 1089115362PRTKluveromyces lactisLeu2Leu2 115Met Ser Lys Asn Ile Val Val Leu Pro Gly Asp His Val Gly Lys Glu1 5 10 15Val Thr Asp Glu Ala Ile Lys Val Leu Asn Ala Ile Ala Glu Val Arg 20 25 30Pro Glu Ile Lys Phe Asn Phe Gln His His Leu Ile Gly Gly Ala Ala 35 40 45Ile Asp Ala Thr Gly Thr Pro Leu Pro Asp Glu Ala Leu Glu Ala Ser 50 55 60Lys Lys Ala Asp Ala Val Leu Leu Gly Ala Val Gly Gly Pro Lys Trp65 70 75 80Gly Thr Gly Ala Val Arg Pro Glu Gln Gly Leu Leu Lys Ile Arg Lys 85 90 95Glu Leu Gly Leu Tyr Ala Asn Leu Arg Pro Cys Asn Phe Ala Ser Asp 100 105 110Ser Leu Leu Asp Leu Ser Pro Leu Lys Pro Glu Tyr Ala Lys Gly Thr 115 120 125Asp Phe Val Val Val Arg Glu Leu Val Gly Gly Ile Tyr Phe Gly Glu 130 135 140Arg Lys Glu Asp Glu Gly Asp Gly Val Ala Trp Asp Ser Glu Lys Tyr145 150 155 160Ser Val Pro Glu Val Gln Arg Ile Thr Arg Met Ala Ala Phe Leu Ala 165 170 175Leu Gln Gln Asn Pro Pro Leu Pro Ile Trp Ser Leu Asp Lys Ala Asn 180 185 190Val Leu Ala Ser Ser Arg Leu Trp Arg Lys Thr Val Glu Glu Thr Ile 195 200 205Lys Thr Glu Phe Pro Gln Leu Thr Val Gln His Gln Leu Ile Asp Ser 210 215 220Ala Ala Met Ile Leu Val Lys Ser Pro Thr Lys Leu Asn Gly Val Val225 230 235 240Ile Thr Asn Asn Met Phe Gly Asp Ile Ile Ser Asp Glu Ala Ser Val 245 250 255Ile Pro Gly Ser Leu Gly Leu Leu Pro Ser Ala Ser Leu Ala Ser Leu 260 265 270Pro Asp Thr Asn Lys Ala Phe Gly Leu Tyr Glu Pro Cys His Gly Ser 275 280 285Ala Pro Asp Leu Pro Ala Asn Lys Val Asn Pro Ile Ala Thr Ile Leu 290 295 300Ser Ala Ala Met Met Leu Lys Leu Ser Leu Asp Leu Val Glu Glu Gly305 310 315 320Arg Ala Leu Glu Glu Ala Val Arg Asn Val Leu Asp Ala Gly Val Arg 325 330 335Thr Gly Asp Leu Gly Gly Ser Asn Ser Thr Thr Glu Val Gly Asp Ala 340 345 350Ile Ala Lys Ala Val Lys Glu Ile Leu Ala 355 360116499DNASaccharomyces cerevisiaepNUP57pNUP57 116tcatctgcgc aatgactatc aagaccttct gcaagaattt caaatctcac tgaaaatctt 60gaccgaaaag tgtcttgaaa acccatcaag cctgcaaaac ctatctttga cattagtctc 120cattataaaa acggcatagt tgggagaaaa cttttcatac ttcaattgtg gactgatata 180agtattttgg ttttgcccgc atgatcatcc cacatggcta cagcagttct ctcataggaa 240atagtacaat agctacgtga tataatctaa ataattgttg ccaatgtgta attatatcat 300tttgaacgtt cgcgaaatgg attattttca aaaattttgt ttcttgaaat gagtaaaagc 360aaaagtccaa ctctccaagt cgatgtaaac aactttttgc caaagggact gaaagactaa 420atcgaggatt atcccgttca aactattcca gaaacgctcg ttagtaacaa aagacatacc 480ttgttgacca attgatcac 499117451DNASaccharomyces cerevisiaepGAP1pGAP1 117cactttcacc agatcccaaa tgtcccgccc ctattcccgt gttccatcac gtaccataac 60ttaccatttc atcacgttct ctatggcaca ctggtactgc ttcgactgct ttgcttcatc 120ttctctatgg gccaatgagc taatgagcac aatgtgctgc gaaataaagg gatatctaat 180ttatattatt acattataat atgtactagt gtggttattg gtaattgtac ttaattttga 240tatataaagg gtggatcttt ttcattttga atcagaattg gaattgcaac ttgtctcttg 300tcactattac ttaatagtaa ttatatttct tattaacctt ttttttaagt caaaacacca 360aggacaagaa ctactcttca aaggtatttc aagttatcat acgtctcaca cacgcttcac 420agtttcaagt aaaaaaaaag aatattacac a 451118998DNASaccharomyces cerevisiaepJEN1pJEN1 118aatgtgttta taaattattt tttttgctgg tagcaaaatc aactcattgt cttccattca 60gagtctaatc gaacgttatc gcaatgcttg cacactttta aacaatacga tttagtttaa 120gtggatggac ccccacgctt agtgttccac aggtttgtcc ccactgtttt tacattccac 180tgtacatttt tgcaatagaa ggtcattgta tgctaccttg ggcggctaag aatacctgta 240aaaatttgga gaaattagat tcgtaaagaa tgactcgcaa cgactccaat gatttcttct 300tttcaccctt tgaacggccg atatccgcgc gggatcctga ccccgcaatt tactccacta 360gaccggcgtg tttctctttt tccttttcct ggggttagag cccaagagct aatagccgac 420aaacggactc caaaaaaaaa aggaggcaca ggacaaacgc agcacctgcg tcattcacgc 480tgaagcggca gcaagcattt tcgatcagct ccaattaaat gaagactatt cgccgtaccg 540ttcccagatg ggtgcgaaag tcagtgatcg aggaagttat tgagcgcgcg gcttgaaact 600atttctccat ctcagagccg ccaagcctac cattattctc caccaggaag ttagtttgta 660agcttctgca caccatccgg acgtccataa ttcttcactt aacggtcttt tgccccccct 720tctactataa tgcattagaa cgttacctgg tcatttggat ggagatctaa gtaacactta 780ctatctccta tggtactatc ctttaccaaa aaaaaaaaaa aaaaaaaaaa aaaaaatcag 840caaagtgaag taccctcttg atgtataaat acattgcaca tcattgttga gaaatagttt 900tggaagttgt ctagtccttc tcccttagat ctaaaaggaa gaagagtaac agtttcaaaa 960gtttttcctc aaagagatta aatactgcta ctgaaaat 998119450DNASaccharomyces cerevisiaepICL1pICL1 119ttttgctact cgtcatccga tgagaaaaac tgttcccttt tgccccaggt ttccattcat 60ccgagcgatc acttatctga cttcgtcact ttttcatttc atccgaaaca atcaaaactg 120aagccaatca ccacaaaatt aacactcaac gtcatctttc actacccttt acagaagaaa 180atatccatag tccggactag catcccagta tgtgactcaa tattggtgca aaagagaaaa 240gcataagtca gtccaaagtc cgcccttaac caggcacatc ggaattcaca aaacgtttct 300ttattatata aaggagctgc ttcactggca aaattcttat tatttgtctt ggcttgctaa 360tttcatctta tccttttttt cttttcacac ccaaatacct aacaattgag agaaaactct 420tagcataaca taacaaaaag tcaacgaaaa 450120598DNASaccharomyces cerevisiaepADH2pADH2 120tatcttaact gatagtttga tcaaaggggc aaaacgtagg ggcaaacaaa cggaaaaatc 60gtttctcaaa ttttctgatg ccaagaactc taaccagtct tatctaaaaa ttgccttatg 120atccgtctct ccggttacag cctgtgtaac tgattaatcc tgcctttcta atcaccattc 180taatgtttta attaagggat tttgtcttca ttaacggctt tcgctcataa aaatgttatg 240acgttttgcc cgcaggcggg aaaccatcca cttcacgaga ctgatctcct ctgccggaac 300accgggcatc tccaacttat aagttggaga aataagagaa tttcagattg agagaatgaa 360aaaaaaaaaa aaaaaaggca gaggagagca tagaaatggg gttcactttt tggtaaagct 420atagcatgcc tatcacatat aaatagagtg ccagtagcga cttttttcac actcgaaata 480ctcttactac tgctctcttg ttgtttttat cacttcttgt ttcttcttgg taaatagaat 540atcaagctac aaaaagcata caatcaacta tcaactatta actatatcgt aatacaca 598121793DNASaccharomyces cerevisiaepMLS1pMLS1 121tgtctaatgc gaaggtactt ttattttttt cagattcaaa gcaatattat ttagacaatt 60gatactaagt gagcttaagg aggattaaac aactgtggaa tccttcacaa ggattcaata 120tttgtttttc ctggttattt tgccatcatt caactttcct cagacgtaaa attcgtgctt 180agtgatgtct caatattccc gcagggtaat aaaattcaat aactatcact atatacgcaa 240cagtattacc ctacattgct atcggctcaa tggaaatccc catatcatag cttccattgg 300gccgatgaag ttagtcgacg gatagaagcg gttgtcccct ttcccggcga gccggcagtc 360gggccgaggt tcggataaat tttgtattgt gttttgattc tgtcatgagt attacttatg 420ttctctttag gtaaccccag gttaatcaat cacagtttca taccggctag tattcaaatt 480atgacttttc ttctgcagtg tcagccttac gacgattatc tatgagcttt gaatatagtt 540tgccgtgatt cgtatcttta attggataat aaaatgcgaa ggatcgatga cccttattat 600tatttttcta cactggctac cgatttaact catcttcttg aaagtatata agtaacagta 660aaatataccg tacttctgct aatgttattt gtcccttatt tttcttttct tgtcttatgc 720tatagtacct aagaataacg actattgttt tgaactaaac aaagtagtaa aagcacataa 780aagaattaag aaa 793

Claims

1. An ectoine-producing recombinant yeast, in the genome of which: (A) (i) at least one nucleic acid encoding an aspartokinase is overexpressed and/or is under the control of an inducible or repressible promoter; and/or (ii) at least one nucleic acid encoding an aspartate kinase is overexpressed and/or is under the control of an inducible or repressible promoter; (B) at least one nucleic acid encoding an aspartate semi-aldehyde dehydrogenase and/or at least one nucleic acid encoding an aspartate semi-aldehyde dehydrogenase that can use as coenzyme both NAD and NADP is overexpressed and/or is under the control of an inducible or repressible promoter; (C) at least one nucleic acid encoding a diaminobutyrate aminotransferase is overexpressed and/or is under the control of an inducible or repressible promoter; (D) (i) at least one nucleic acid encoding an homoserine-O-acetyltransferase MET2 is overexpressed and/or is under the control of an inducible or repressible promoter; (ii) at least one nucleic acid encoding an homoserine-O-acetyltransferase METX is overexpressed and/or is under the control of an inducible or repressible promoter, and/or (iii) at least one nucleic acid encoding a diaminobutyric acid acetyltransferase is overexpressed and/or is under the control of an inducible or repressible promoter; (E) at least one nucleic acid encoding an ectoine synthase is overexpressed and/or is under the control of an inducible or repressible promoter; (F) (i) at least one endogenous nucleic acid encoding an homoserine dehydrogenase has been deleted and/or interrupted, and/or (ii) at least one nucleic acid encoding an homoserine dehydrogenase is independently: under the control of an inducible or repressible promoter; under the control of a weak promoter; and/or in a destabilized form.

2. The recombinant yeast according to claim 1, in the genome of which at least one nucleic acid encoding an aspartate transaminase is overexpressed and/or is under the control of an inducible or repressible promoter.

3. The recombinant yeast according to claim 1, in the genome of which at least one nucleic acid encoding a glutamate dehydrogenase that converts oxo-glutarate to glutamate is overexpressed and/or is under the control of an inducible or repressible promoter.

4. The recombinant yeast according to claim 1, in the genome of which at least one of the following modifications has been performed: (A) at least one endogenous nucleic acid encoding a general amino acid permease AGP3 have been deleted from the genome of the yeast, and: (i) at least one nucleic acid encoding a general amino acid permease AGP3 has been inserted and is under the control of an inducible or repressible promoter, and/or (ii) at least one nucleic acid encoding a destabilized general amino acid permease AGP3 has been inserted; (B) at least one endogenous nucleic acid encoding a branched-chain amino-acid permease 3 has been deleted from the genome of the yeast, and: (i) at least one nucleic acid encoding a branched-chain amino-acid permease 3 has been inserted and is under the control of an inducible or repressible promoter, and/or (ii) at least one nucleic acid encoding a destabilized branched-chain amino-acid permease 3 has been inserted; (C) at least one endogenous nucleic acid encoding a branched-chain amino-acid permease 2 has been deleted from the genome of the yeast, and: (i) at least one nucleic acid encoding a branched-chain amino-acid permease 2 has been inserted and is under the control of an inducible or repressible promoter, and/or (ii) at least one nucleic acid encoding a destabilized branched-chain amino-acid permease 2 has been inserted; (D) at least one endogenous nucleic acid encoding a general amino acid permease GAP1 has been deleted from the genome of the yeast, and: (i) at least one nucleic acid encoding a general amino acid permease GAP1 has been inserted and is under the control of an inducible or repressible promoter, and/or (ii) at least one nucleic acid encoding a destabilized general amino acid permease GAP1 has been inserted; (E) at least one endogenous nucleic acid encoding a high-affinity glutamine permease GNP1 has been deleted from the genome of the yeast, and: (i) at least one nucleic acid encoding a high-affinity glutamine permease GNP1 has been inserted and is under the control of an inducible or repressible promoter, and/or (ii) at least one nucleic acid encoding a destabilized high-affinity glutamine permease GNP1 has been inserted; (F) at least one endogenous nucleic acid encoding a general amino acid permease AGP1 has been deleted from the genome of the yeast, and: (i) at least one nucleic acid encoding a general amino acid permease AGP1 has been inserted and is under the control of an inducible or repressible promoter, and/or (ii) at least one nucleic acid encoding a destabilized general amino acid permease AGP1 has been inserted; (G) at least one endogenous nucleic acid encoding a low-affinity methionine permease MUP3 has been deleted from the genome of the yeast, and: (i) at least one nucleic acid encoding a low-affinity methionine permease MUP3 has been inserted and is under the control of an inducible or repressible promoter, and/or (ii) at least one nucleic acid encoding a destabilized low-affinity methionine permease MUP3 has been inserted; (H) at least one endogenous nucleic acid encoding a high-affinity methionine permease MUP1 has been deleted from the genome of the yeast, and: (i) at least one nucleic acid encoding a high-affinity methionine permease MUP1 has been inserted and is under the control of an inducible or repressible promoter, and/or (ii) at least one nucleic acid encoding a destabilized high-affinity methionine permease MUP1 has been inserted; (I) at least one nucleic acid encoding a probable transporter AQR1 is overexpressed; and/or (J) at least one nucleic acid encoding a polyamine transporter 1 is overexpressed.

5. The recombinant yeast according to claim 4, in the genome of which at least two of the modifications indicated in claim 4 have been performed.

6. The recombinant yeast according to claim 1, wherein the nucleic acid encoding an aspartokinase are nucleic acid from a yeast.

7. The recombinant yeast according to claim 1, wherein the nucleic acid encoding an homoserine-O-acetyltransferase METX are nucleic acid from a bacterium.

8. The recombinant yeast according to claim 1, wherein the at least one nucleic acid encoding a general amino acid permease, a branched-chain amino-acid permease 3, a branched-chain amino-acid permease 2, a general amino acid permease GAP1, a high-affinity glutamine permease GNP1, a general amino acid permease AGP1, a low-affinity methionine permease MUP3 and a high-affinity methionine permease MUP1 are, independently, nucleic acid from a yeast.

9. The recombinant yeast according to claim 1, wherein at least one of the overexpressed nucleic acid is under the control of a strong promoter.

10. The recombinant yeast according to claim 1, wherein the inducible or repressible promoter is, independently, selected from the group consisting of promoters inducible or repressible with copper, promoters inducible or repressible with methionine and promoters inducible or repressible with threonine.

11. The recombinant yeast according to claim 1, wherein the weak promoter is, independently, selected from the group consisting of pURA3, pRPLA1, pNUP57 and pGAP1.

12. The recombinant yeast according to claim 1, wherein the inducible or repressible promoter is, independently, selected from the group consisting of promoters inducible or repressible with copper, promoters inducible or repressible with lysine and promoters inducible or repressible with methionine.

13. Method for producing ectoine, said method comprising the steps of: (a) culturing a recombinant yeast as defined in claim 1 in a culture medium; and (b) recovering the ectoine from said culture medium.

14. Method according to claim 13, wherein the culture medium comprises at least a carbon source.

15. (canceled)